Methods for controlling amplification

ABSTRACT

Methods of amplifying nucleic acid on a solid support are described. Beads and template, each in known concentrations, are employed so a range of template to bead ratios can be exploited. Where the beads contain primers, the template can be amplified. After amplification, non-covalently bound template is removed, so as to leave beads with extended primers (or beads with primers that were not extended).

FIELD OF THE INVENTION

The present invention relates to methods and compositions for theproduction of biomolecules on beads or particles, for example byamplification or de novo synthesis (e.g. by enzymatically mediatedreplication or enzymatically mediated synthesis, respectively). Thisinvention also relates to methods and compositions for thephoto-transfer of substances and compounds, such as biomolecules, fromone surface to another. This invention has applications in many fieldsincluding, but not limited to, the fields of microarrays and micro-beadtechnologies, for applications such as parallel DNA sequencing, mRNA orprotein expression profiling, single nucleotide polymorphism (SNP) andother genetic analyses, biomarker discovery, diagnostics, prognostics,personalized medicine, protein interaction analysis, drug discovery andproteomics.

BACKGROUND OF THE INVENTION

Microarray and micro-bead technologies can be used as tools to conductbiological, chemical or biochemical analyses in a parallel, massivelyparallel or multiplexed fashion because of the large number of differentcompounds or substances that can be fabricated or deposited on themicroarray substrate or beads. As is also well known in the art,microarrays and micro-bead technologies are applicable to a variety ofsuch analyses including, but not limited to, mRNA or protein expressionprofiling, parallel DNA sequencing, protein-protein interaction mapping,protein-drug interaction analysis, antibody specificity testing, enzymesubstrate profiling and single nucleotide polymorphism (SNP) detectionas well as various other applications in the fields of biomarkerdiscovery, diagnostics, prognostics, personalized medicine, proteininteraction analysis, drug discovery and proteomics (See for example[Ramachandran et al. (2004) Science 305, 86-90; Zhu et al. (2001)Science 293, 2101-2105; MacBeath & Schreiber. (2000) Science 289,1760-1763; Zhu et al. (2000) Nat Genet. 26, 283-289; Michaud et al.(2003) Nat Biotechnol 21, 1509-1512; Sheridan. (2005) Nat Biotechnol 23,3-4; Robinson et al. (2003) Nat Biotechnol 21, 1033-1039; Robinson etal. (2002) Nat Med 8, 295-301; Xiao et al. (2007) Bioimformatics 23,1459-1467; Hughes et al. (2007) Anticancer Res 27, 1353-1359]).

The plurality of compounds or substances arrayed or displayed on themicroarray substrate or micro-beads can be of a variety of types and fora variety of uses. These compounds or substances are not intended to belimited to any one type or for any one use, and henceforth will bereferred to “features”, as is commonly used in the art of microarrays.Microarray or micro-bead features can include, but are not limited toproteins, peptides, DNA, nucleic acids, nucleosides, nucleotides orpolymers thereof, drug or other chemical compounds, polymers, cells,tissues, particles, nanoparticles or nanocrystals. Microarray ormicro-bead features may be used as, for example, analytes, probes ortargets in various applications, assays or analyses.

Microarrays currently exist as two-dimensional feature arrays fabricatedon solid glass (plain or chemically activated/modified) or nylonsubstrates for instance. A variety of additional substrates such asnitrocellulose, polystyrene, polymeric or metallic materials provided assolid substrates, coatings, films, membranes or matrices are alsoavailable. Due to the massively parallel or multiplexed nature ofmicroarrays, far more information is obtained from a single experimentcompared to other non-parallel or non-multiplexed methods. Furthermore,because the samples to be analyzed are generally in limited supply, hardto produce and/or expensive, it is highly desirable to performexperiments on as many components in a mixture as possible on as manyfeatures as possible, on a single microarray. This calls for asignificant increase in feature density and quantity on a singlesubstrate. In general, microarrays with densities larger than 400features per square centimeter are referred as “high density”microarrays, otherwise, they are “low density” microarrays. AffymetrixInc. (Santa Clara, Calif.) for example, currently offers severalcommercial high density oligonucleotide microarrays having as much as 1million or more ˜10 μm features, for feature densities reaching ˜1million/cm² [Barone et al. (2001) Nucleosides Nucleotides Nucleic Acids20, 525-531]. Applications of these commercial microarrays include mRNAexpression profiling or single nucleotide polymorphism (SNP) detection.

Production of microarray or micro-bead features can be achieved by avariety of methods, either by in situ production, or bydeposition/binding of off-line produced feature substances ontomicroarray substrates, beads or particles. Current methods however,suffer from various deficiencies.

For microarrays, there are two categories of techniques on the market,photolithographic and mechanical printing. Photolithography is an insitu method, while mechanical printing techniques require off-lineproduction of the feature substances followed by deposition of thefeatures onto the microarray substrate. The photolithographic techniqueadapts the same fabrication process used for electronic integratedcircuits, in order to in situ synthesize compounds or substances,monomer-by-monomer for example (e.g. nucleic acid monomers), directly onthe microarray substrate. This technique requires a large capital outlayfor equipment, running up to hundreds of millions of dollars. Theinitial setup of new microarray designs is also very expensive due tothe high cost of producing photo masks. This technique is therefore onlyviable in mass production of standard microarrays at a very high volume.Even at high volumes, the complexity in synthesis still limits theproduction throughput resulting in a high microarray cost. This methodhas typically been employed for high density DNA microarrays. Thecomplexity of the process however, also limits the length of thesynthesized DNA to the level of short oligonucleotide sequences of about25 bases.

The established mechanical printing technique [U.S. Pat. No. 5,807,522]uses a specially designed mechanical robot, which produces a featurespot on the microarray by dipping a pin head into a fluid, i.e. the bulkstocks of the feature substances, such as DNA or protein solutions, andthen printing it onto the substrate at a predetermined position. Washingand drying of the pins are required prior to printing a differentfeature onto the microarray substrate. In current designs of suchrobotic systems, the printing pin, and/or the stage carrying themicroarray substrates move along the XYZ axes in coordination to depositsamples at controlled positions on the substrates. Other mechanicalprinting techniques, either contact or non-contact, use quills, pinswith built-in sample channels, non-contact ink jet/piezoelectric devicesor capillaries as the means of feature deposition. Because a microarraycontains a very large number of different features, these techniques,although highly flexible, are inherently very slow. Even though thespeed can be enhanced by employing multiple pin-heads (or printingdevices) and printing multiple substrates before washing, productionthroughput remains very low. Furthermore, the printing instrumentationis susceptible to mechanical failure due to the large number of movingparts. Non-contact methods additionally suffer from difficulties incontrolling the microarray quality. Mechanical printing methods aretherefore not suitable for high volume mass production of microarrays.

Mechanical printing also requires that the materials comprising thefeatures be produced off-line, prior to printing. Typically, bulk stocksof the feature substances are produced and used to print multiple spotsand/or microarrays. However, such production has a variety oflimitations. For example, conventional off-line production of DNA (e.g.oligonucleotides) uses chemical synthesis, but is limited toapproximately 150 bases in length, and although can be done in parallel,is not truly multiplexed. Conventional methods for DNA production beyondthis length (e.g. full-length genes or large portions thereof), involvesslow, laborious, and non-multiplexed standard DNA cloning practices.Adams and Kron [U.S. Pat. No. 5,641,658] disclose a general multiplexedmethod for producing DNA on beads or other surfaces by using solid-phasebridge PCR (i.e. where both PCR primers, forward and reverse, areattached to the surface). However, this approach is rarely used and hasnot been adapted for cloning (amplification of single templatemolecules) or downstream production of protein, for example. Forrecombinant proteins for instance, off-line production typicallyinvolves all the aforementioned conventional DNA cloning procedures inaddition to labor intensive and non-multiplexed steps such astransfection, cell culture and purification reactions for each proteinspecies. It is particularly important yet challenging to deposit theproduced feature substances in pure and active form on the microarraysubstrate. Prior to deposition, feature substances are usually producedin heterogeneous mixtures and hence require purification. Theproduction, purification and deposition process can readily inactivatedelicate feature substances such as proteins. Furthermore, contaminantson the microarray surface can yield false signals in downstreamanalyses.

Feature size is another limiting factor of high density microarrayproduction. With either microarray fabrication technique,photolithography or mechanical printing, the microarrays cannot easilybe extended to spot sizes (i.e. features) at the nanometer level. Suchnanoarrays would be highly advantageous, since they could dramaticallyincrease the level of multiplexing for example. Photolithographyrepresents the state-of-the-art in terms of spot size (10 μm) anddensity, but is limited to short polymers such as oligonucleotides andshort peptides, and is essentially only used in practice for DNAmicroarrays.

Micro-bead technologies are analogous to microarrays except that thefeatures are spatially segregated on different beads or particles. Theexperiment, analysis and/or readout can be formatted like a microarray,for example, with the beads arrayed or embedded on the surface or inwells of a device such as a microscope slide or plate. The experiment,analysis and/or readout can alternatively be performed with the beadssuspended in a solution for example. The working density of features formicro-bead technologies is potentially far greater than for microarrays,depending primarily on the minimum usable bead size and maximum usablebead concentration or density. For example, 0.3 μm beads have beenarrayed in etched wells at densities of 4×10⁹ beads/cm² [Michael et al.(1998) Anal Chem 70, 1242-1248], three orders of magnitude better thanthe current high density DNA microarrays from Affymetrix Inc. (SantaClara, Calif.). However, because the beads are random, a decoding methodis typically required to determine the identity of the feature on eachbead in a given experiment or analysis. Several commercial entitiesutilize micro-bead technologies to achieve parallel or multiplexedassays in a fashion similar to microarrays. For example, LuminexCorporation (Austin, Tex.) markets a flow cytometry based bead platformfor multiplexed assays, such as SNP detection and various immunoassays.Beads are fluorescently coded to facilitate the multiplexing andproduction of the bead “features”, e.g. analytes, is up to the end-user.Illumina Incorporated (San Diego, Calif.) has created a bar-codedbead-array platform for genetic analyses, such as multiplexed SNP andDNA methylation detection. 454 Life Sciences™ (Branford, Conn.) offers abead-based parallel sequencing platform whereby beads carrying the DNA“features”, in this case DNA analytes for sequencing, are arrayed inmicroscopic wells and analyzed by massively parallel DNA pyrosequencing,for applications such as whole genome sequencing and detection of lowabundance mutations.

In general, production of a plurality of beads with different features,whether the features are to serve as probes, targets or analytes forexample, suffers from analogous problems as described for microarrays.For instance, different feature substances are typically producedoff-line and can then be bound to beads in separate reactors, in amechanical process of mixing solutions containing the feature substanceswith beads containing some binding capacity. This can be done inseparate test tubes, vials or wells of a microtiter plate for example.Liquid handling robotics may be used to perform this process inparallel, however, it is again not truly multiplexed (e.g. does notproduce the complete population of beads with different features, usinga single reaction or few reactions within a single reactor).

The present invention overcomes the problems and disadvantagesassociated with current strategies and designs for the fabrication andutilization of microarrays, micro-bead technologies and a variety ofother parallel, massively parallel or multiplexed biological sensingmethodologies or devices.

SUMMARY OF THE INVENTION

The present invention relates to methods and compositions for theproduction of biomolecules on beads or particles, for example byamplification or de novo synthesis (e.g. by enzymatically mediatedreplication or enzymatically mediated synthesis, respectively). Thisinvention also relates to methods and compositions for the transfer(e.g. photo-transfer) of substances and compounds, such as biomolecules,from one surface to another. This invention has applications in manyfields including, but not limited to, the fields of microarrays andmicro-bead technologies, for applications such as parallel DNAsequencing, mRNA or protein expression profiling, single nucleotidepolymorphism (SNP) and other genetic analyses, biomarker discovery,diagnostics, prognostics, personalized medicine, protein interactionanalysis, drug discovery and proteomics.

In one embodiment, the present invention contemplates transferringcompounds from one surface to another. While it is not intended that thepresent invention be limited by the nature of the compound (e.g. drug,ligand, etc.), preferred compounds are biomolecules (e.g. proteins,protein fragments, peptides, nucleic acid, oligonucleotides, etc.). Inone embodiment, the present invention contemplates a method fortransferring a compound from a first surface to a second surface,comprising: a) providing i) a compound attached to a first surfacethrough a photocleavable linker; ii) a source of electromagneticradiation; and iii) a second surface; b) contacting said second surfacewith said compound; and c) illuminating said compound with radiationfrom said radiation source under conditions such that compound isphotocleaved from said first surface and transferred to said secondsurface. Electromagnetic radiation includes x-rays, ultraviolet rays,visible light, infrared rays, microwaves, radio waves, and combinationsthereof.

In one embodiment, said first surface is part of a particle. In apreferred embodiment, said particle is a bead and said contactingcomprising depositing said bead onto said second surface. In a preferredembodiment, the method further comprises, after step c), the step d)removing said bead(s) from said second surface. Of course, theefficiency of removing the beads need not be 100%; some beads(preferably less than 50%, more preferably less than 20%, and mostpreferably not more than 1%) may remain after the removing step.

In some embodiments, it is not strictly necessary that said compound bein physical contact with said second surface. Without limiting thepresent invention to any particular mechanism, it is believed that it issufficient that the compound be in proximity (e.g. to a distance of lessthan 106 Angstroms, more preferably between 0.1 and 1000 Angstroms) tosaid second surface. In one embodiment, the compound is brought intoproximity simply by bringing the surfaces into proximity (without actualcontact between the surfaces). In one embodiment, the compound isbrought into proximity via a carrier, such as a particle or bead. Forexample, the present invention, in one preferred embodiment,contemplates a method for transferring substances from a bead to asurface, comprising: a) providing i) a compound attached to a beadthrough a photocleavable linker; ii) a source of electromagneticradiation; and iii) a surface; b) bringing said bead into contact with(or in proximity to) said surface; and illuminating said bead withradiation from said radiation source under conditions such that compoundis photocleaved from said bead and transferred to said surface. In oneembodiment, step b) comprises depositing said bead onto said surface(whether by hand or by robotic spotting or by inkjet spraying or bysedimentation or the like).

It is not intended that the present invention be limited to particularsurfaces. In one embodiment, the surface is part of a solid support. Forexample, in one embodiment said first surface is part of a particle ornanoparticle. In one embodiment, the particle is a bead. In oneembodiment, said nanoparticle is a nanocrystal. Indeed, surfaces can bebeads, glass slides and surfaces used for biomolecular detection (e.g.surfaces used for mass spec). In one embodiment, said second surface isselected from glass surfaces, metal surfaces, surfaces coated withantibodies, surfaces coated with streptavidin, surfaces coated withcells, hydrogel surfaces, nitrocellulose surfaces, polymeric surfaces,gold coated surfaces, surfaces suitable for surface plasmon resonance,surfaces suitable for MALDI, surfaces coated with nucleic acid, andsurfaces coated with protein.

It is not intended that the present invention be limited by the natureof the surfaces employed. A variety of surface types (e.g. coated,charged, absorbing, non-absorbing, etc.) and surface configurations(e.g. flat, curved, indented, etc.) are contemplated. Where coatedsurfaces are used, the surface may be coated with a variety of molecules(whether nucleic acids, proteins, carbohydrates or other types). In apreferred embodiment, the surface is coated with molecules havingaffinity to another molecule (e.g. binding partner) such as antibodies,lectins, avidin, streptavidin, and the like. In some embodiments, thesurface is coated with a metal (such as gold, platinum, copper, etc.) ormetal ions. In some embodiments, metal ion-chelate derivatives, nickelnitrilo-triacetic acid (Ni-NTA), or cobalt nitrilo-triacetic acidcomplexes are employed. In some embodiments, said surfaces are coatedwith cells. Surface types such as hydrated matrix coated surfaces (e.g.polyacrylamide gels or HydroGel coated microarray substrates;PerkinElmer Life and Analytical Sciences, Inc., Boston, Mass.),nitrocellulose surfaces, polymeric surfaces, surfaces suitable forsurface plasmon resonance, and surfaces suitable for mass spectrometry(e.g. MALDI) are specifically contemplated. In some embodiments, polymersurfaces are used (e.g. polyvinylidene fluoride (PVDF) or polystyrene).In other embodiments, plastic or ceramic surfaces are used. In someembodiments, simple surfaces such as glass, crystal, and silicon dioxidesurfaces are used (e.g. in one embodiment of the above described method,said second surface may be a glass surface). In some embodiments, thesurface is modified with a compound to make the surface morehydrophilic. Rain-X antifog (commercially available) is a surfacetreatment which makes surfaces hydrophilic. Hydrophobic surfaces (suchas Teflon) can also be employed. In some embodiments, the surface ismodified with a magnetic or paramagnetic coating. In some embodiments,the surface is modified so as to comprise reactive groups (e.g. aminereactive groups, esters, epoxy groups, etc.).

In one embodiment, said second surface is selected from glass surfaces,metal surfaces, surfaces coated with antibodies, surfaces coated withstreptavidin, surfaces coated with cells, hydrogel surfaces,nitrocellulose surfaces, polymeric surfaces, gold coated surfaces,surfaces suitable for surface plasmon resonance, surfaces suitable forMALDI, surfaces coated with nucleic acid, and surfaces coated withprotein.

Where beads are used, it is not intended that the present invention belimited to the particular type. A variety of bead types are commerciallyavailable, including but not limited to, beads selected from agarosebeads, streptavidin-coated beads, NeutrAvidin-coated beads,antibody-coated beads, paramagnetic beads, magnetic beads, electrostaticbeads, electrically conducting beads, fluorescently labeled beads,colloidal beads, glass beads, semiconductor beads, and polymeric beads.

Importantly, a variety of compounds can be photo-transferred using themethods of the present invention, including but not limited to compoundsselected from the group consisting of proteins, peptides, antibodies,amino acids, amino acid analogs, drug compounds, nucleic acids,nucleosides, nucleotides, lipids, fatty acids, saccharides,polysaccharides, inorganic molecules, and metals. In one embodiment,said compound is selected from the group consisting of proteins, nascentproteins, peptides, antibodies, amino acids, amino acid analogs, drugcompounds, nucleic acids, nucleosides, nucleotides, protein-nucleic acidcomplexes, lipids, fatty acids, saccharides, polysaccharides, inorganicmolecules, and metals. In one embodiment, the compound is a conjugatecomprising two or more different molecules. For example, in oneembodiment, said conjugate comprises an antibody-protein complex, and inparticular, and antibody-nascent protein complex.

Photocleavage of the photoconjugate may cause the compound or compoundsto be released in a modified or unmodified form. For example, thephotocleavage may leave part of the linker attached to the compound.

It is not intended that the present invention be limited to particularphotocleavable linkers. There are a variety of known photocleavablelinkers. Preferred comprise a 2-nitrobenzyl moiety. U.S. Pat. No.5,643,722 describes a variety of such linkers and is hereby incorporatedby reference.

In one embodiment, the present invention contemplates a method fortransferring substances from a bead to a surface, comprising: a)providing i) a compound attached to a bead through a photocleavablelinker; ii) a source of electromagnetic radiation; and iii) a surface;b) bringing said bead into contact (or in proximity to) with saidsurface; and c) illuminating said bead with radiation from saidradiation source under conditions such that compound is photocleavedfrom said bead and transferred to said surface. In one embodiment, stepb) comprises depositing said bead onto said surface. In a preferredembodiment, the method further comprises, after step c), the step d)removing said bead from said surface. Again, not all beads need beremoved; some (e.g. 1-10%) can remain after washing. Again, the bead canbe of any type (see above) and the compound can be of any type,including biomolecules (see above) and conjugates of biomolecules.

In one embodiment, the method also involves the use of a coding agent(discussed in more detail below). For example, in one embodiment, thepresent invention contemplates a method for transferring substances froma bead to a surface, comprising: a) providing i) a compound attached toa bead through a photocleavable linker, said bead further comprising acoding agent attached to said bead through a photocleavable linker; ii)a source of electromagnetic radiation; and iii) a surface; b) bringingsaid bead into contact with said surface; and c) illuminating said beadwith radiation from said radiation source under conditions such thatsaid compound and said coding agent are photocleaved from said bead andtransferred to said surface. In one embodiment, step b) comprisesdepositing said bead onto said surface. In a preferred embodiment, themethod further comprises, after step c), the step d) removing said beadfrom said surface. Again, not all beads need be removed; some (e.g.1-10%) can remain after washing. Again, the bead can be of any type (seeabove) and the compound can be of any type, including biomolecules (seeabove) and conjugates of biomolecules. In one embodiment, the methodfurther comprises, after step d), the step e) using said coding agent todetermine the identity of said compound.

It is not intended that the present invention be limited to the natureof the coding agent. In one embodiment, said coding agent is selectedfrom the group consisting of nucleic acid, protein, nanoparticles,quantum dots, mass coding agents, and fluorescent molecules. In oneembodiment, the coding agent has identifiable spectral properties andthe identifiable spectral property is detecting using a method selectedfrom fluorescence spectroscopy, absorption spectroscopy, infraredspectroscopy, Raman spectroscopy, nuclear magnetic resonance, massspectrometry.

As mentioned above, the compound can be a ligand. In one embodiment, thepresent invention contemplates a method for transferring a compound froma bead to a surface, comprising: a) providing i) a photocleavablebiotin-labeled compound attached to an avidin-coated bead; ii) a sourceof electromagnetic radiation; and iii) a surface; b) bringing said beadinto contact with (or in proximity to) said surface; and c) illuminatingsaid bead with radiation from said radiation source under conditionssuch that compound is photocleaved from said bead and transferred tosaid surface. In a preferred embodiment, the method further comprises,after step c), the step d) removing said bead from said surface. Again,not all beads need be removed; some (e.g. 1-10%) can remain afterwashing. Again, the bead can be of any type (see above) and the compoundcan be of any type, including biomolecules (see above) and conjugates ofbiomolecules. In one embodiment, said photocleavable biotin comprises a2-nitrobenzyl moiety. In one embodiment, said compound is a nascentprotein labeled with photocleavable biotin during translation.

The transfer (e.g. photo-transfer) of compounds and substances has manyuses, including but not limited to, the formation of arrays. Forexample, in one embodiment, the present invention contemplates a methodof making an array, comprising: a) providing i) a plurality of beads (orother particles or nanoparticles), each bead comprising a group ofcompounds, each compound of said group attached to a bead through aphotocleavable linker; ii) a source of electromagnetic radiation; andiii) a surface; b) bringing said plurality of beads into contact with(or in proximity to) said surface; and c) illuminating said beads withradiation from said radiation source under conditions such that at leasta portion of said compounds is photocleaved from said beads andtransferred to said surface to form a plurality of transferred groups ofcompounds on said surface, at least a portion of said plurality oftransferred groups positioned in different locations on said surface (soas to create an array). In one embodiment, said photocleavable linkercomprises a 2-nitrobenzyl moiety.

It is not intended that the present invention be limited to the natureof compounds employed or the makeup of compounds within a group. Forexample, the present invention contemplates an embodiment wherein eachcompound in any one group of compounds of step a) is identical. Inanother embodiment, two or more different compounds are in a group. Inanother embodiment, each transferred group of step c) has fewercompounds than any one group of compounds of step a). In anotherembodiment, at least a portion of said plurality of transferred groupsare positioned at different predetermined locations on said surface. Inanother embodiment, step b) comprises depositing single beads atdifferent locations on said surface. In another embodiment, step b)comprises depositing more than one bead at each location on saidsurface. In a preferred embodiment, the method further comprises, afterstep c), the step d) removing at least a portion of said beads from saidsurface (some, e.g. 1-10%, of the beads can remain). All types ofcompounds are contemplated, including biomolecules. For example, in oneembodiment, each compound of every transferred group is a peptide, andin particular, a peptide of between 6 and 50 amino acids in length. Inanother, embodiment, each compound of every transferred group is anoligonucleotide, and in particular, an oligonucleotide of between 18 and150 nucleotides in length. In one embodiment, each compound of everytransferred group comprises nucleic acid derived from a single nucleicacid template. In one embodiment, each group consists of the amplifiedproduct from a single nucleic acid template.

As noted above, coding agents can be employed in the methods of thepresent invention. Coding agents are particularly useful in the contextof arrays, and in particular, random arrays (in a way, ordered arraysare already coded by position, e.g. a compounds x-y location on thesurface). In one embodiment, the present invention contemplates a methodof making an array, comprising: a) providing i) a plurality of beads (orother particles or nanoparticles), each bead comprising a group ofcompounds and at least one coding agent, each compound of said groupattached to a bead through a photocleavable linker, said coding agentattached to said bead through a photocleavable linker; ii) a source ofelectromagnetic radiation; and iii) a surface; b) bringing saidplurality of beads into contact with said surface; and c) illuminatingsaid beads with radiation from said radiation source under conditionssuch that at least a portion of said compounds is photocleaved from saidbeads and transferred to said surface to form a plurality of transferredgroups of compounds on said surface (so as to create an array), at leasta portion of said plurality of transferred groups positioned indifferent locations on said surface and associated with a transferredcoding agent. In one embodiment, the method further comprises, afterstep d), the step e) using said coding agent to determine the identityof said compounds in said portion of said transferred groups. In oneembodiment, each compound in any one group of compounds of step a) isidentical. In another embodiment, two or more different compounds are ina group. In another embodiment, each transferred group of step c) hasfewer compounds than any one group of compounds of step a). In anotherembodiment, at least a portion of said plurality of transferred groupsare positioned at different predetermined locations on said surface. Inanother embodiment, step b) comprises depositing single beads atdifferent locations on said surface. In another embodiment, step b)comprises depositing more than one bead at each location on saidsurface. In a preferred embodiment, the method further comprises afterstep c), the step d) removing at least a portion of said beads from saidsurface (some, e.g. 1-10%, of the beads can remain). All types ofcompounds are contemplated, including biomolecules. For example, in oneembodiment, each compound of every transferred group is a peptide, andin particular, a peptide of between 6 and 50 amino acids in length. Inanother, embodiment, each compound of every transferred group is anoligonucleotide, and in particular, an oligonucleotide of between 18 and150 nucleotides in length. In one embodiment, each compound of everytransferred group comprises nucleic acid derived from a single nucleicacid template. In one embodiment, each group consists of the amplifiedproduct from a single nucleic acid template.

It is not intended that the present invention be limited by the natureof the particle or bead. In one embodiment, the bead is selected fromagarose beads, streptavidin-coated beads, NeutrAvidin-coated beads,antibody-coated beads, paramagnetic beads, magnetic beads, electrostaticbeads, electrically conducting beads, fluorescently labeled beads,colloidal beads, glass beads, semiconductor beads, nanocrystalline beadsand polymeric beads.

It is not intended that the present invention be limited by the natureof the surface. In one embodiment, said surface is selected from chargedsurfaces, hydrophobic surfaces, and hydrophilic surfaces. In oneembodiment, said surface is a chemically treated surface. In oneembodiment, said surface is an epoxy-activated surface. In oneembodiment, said surface is selected from surfaces coated withantibodies, surfaces coated with streptavidin, surfaces coated withcells, surfaces coated with nucleic acid, and surfaces coated withprotein. In one embodiment, said surface is selected from glasssurfaces, hydrogel surfaces, nitrocellulose surfaces, polymericsurfaces, gold coated surfaces, surfaces suitable for surface plasmonresonance, and surfaces suitable for MALDI. On the other hand,embodiments are also contemplated wherein said surface is an untreatedsurface. In one embodiment, said untreated surface is a polymer. In oneembodiment, said polymer is selected from the group consisting ofpolystyrene and polyvinylidene fluoride.

In another embodiment, the present invention contemplates a method ofmaking an array, comprising: providing i) a plurality of avidin-coatedbeads (or other particles or nanoparticles), each bead comprising agroup of photocleavable biotin-labeled compounds attached to said beadthrough a biotin-avidin attachment; ii) a source of electromagneticradiation; and iii) a surface; bringing said plurality of beads intocontact with said surface; and illuminating said beads with radiationfrom said radiation source under conditions such that at least a portionof said compounds is photocleaved from said beads and transferred tosaid surface to form a plurality of transferred groups of compounds onsaid surface, at least a portion of said plurality of transferred groupspositioned in different locations on said surface. In one embodiment,each compound in any one group of compounds of step a) is identical. Inanother embodiment, two or more different compounds are in a group. Inanother embodiment, each transferred group of step c) has fewercompounds than any one group of compounds of step a). In anotherembodiment, at least a portion of said plurality of transferred groupsare positioned at different predetermined locations on said surface. Inanother embodiment, step b) comprises depositing single beads atdifferent locations on said surface. In another embodiment, step b)comprises depositing more than one bead at each location on saidsurface. In a preferred embodiment, the method further comprises, afterstep c), the step d) removing at least a portion of said beads from saidsurface (some, e.g. 1-10%, of the beads can remain). All types ofcompounds are contemplated, including biomolecules. For example, in oneembodiment, each compound of every transferred group is a peptide, andin particular, a peptide of between 6 and 50 amino acids in length. Inanother, embodiment, each compound of every transferred group is anoligonucleotide, and in particular, an oligonucleotide of between 18 and150 nucleotides in length. In one embodiment, each compound of everytransferred group comprises nucleic acid derived from a single nucleicacid template. In one embodiment, each group consists of the amplifiedproduct from a single nucleic acid template.

In some embodiments, amplification on a solid support (such as a bead orother particle) is useful for particular templates, including treatedtemplates (e.g. treated enzymatically, chemically, etc.). In oneembodiment, the present invention contemplates a method of amplifyingnucleic acid on a solid support, comprising: a) providing a populationof beads, each bead comprising one or more amplification primers, apopulation of nucleic acid template molecules, wherein said nucleic acidtemplate molecules have been treated with bisulfite; and b) contactingsaid population of beads with said population of nucleic acid templatemolecules under conditions such that at least a portion of said nucleicacid is amplified to create loaded beads comprising immobilizedamplified nucleic acid. In one embodiment, the method further comprises:c) treating said immobilized amplified nucleic acid so as to release atleast a portion from said loaded beads so as to create free amplifiednucleic acid. In one embodiment, the method further comprises: c)transferring at least a portion of said immobilized amplified nucleicacid to a non-bead solid support. In one embodiment, the method furthercomprises: c) detecting at least a portion of said immobilized amplifiednucleic acid. In one embodiment, the method further comprises: c)determining at least a portion of the sequence of the immobilizedamplified nucleic acid on one or more beads. In one embodiment,determining at least a portion of the sequence comprises use of nucleicacid hybridization probes, single base extension, DNA sequencing andmass spectrometry (or other assay). In one embodiment, the methodfurther comprises: c) transcribing and translating the immobilizedamplified nucleic acid. In one embodiment, each bead of step (a)comprises a forward and a reverse PCR primer. While it is not intendedthat the present invention be limited to particular chemistries, in oneembodiment, prior to step (a) said forward and reverse PCR primerscomprised 5′ amine modifications and were attached to agarose beadscomprising a plurality of primary amine reactive functional groups. Itis not intended that the present invention be limited to the nature ofthe primers. In one embodiment, the primers have a region ofcomplementarity to a gene associated with methylation and/or methylationdifferences associated with disease. In one embodiment, said forward andreverse PCR primers have a region that is completely complementary to aportion of the vimentin gene. In another embodiment, said forward andreverse PCR primers have a region that is completely complementary to aportion of the RASF2A gene. In one embodiment, said forward primercomprises a portion encoding an N-terminal epitope tag and said reverseprimer comprises a portion encoding a C-terminal epitope tag. In someembodiments, the amount of beads and template is known. For example, inone embodiment, the known number of beads and the known number ofnucleic acid template molecules is such that less than five templatemolecules contact any one bead. In another embodiment, the known numberof beads and the known number of nucleic acid template molecules is suchthat less than two template molecules contact any one bead. In stillanother embodiment, the known number of beads and the known number ofnucleic acid template molecules is such that less than one templatemolecule contacts any one bead. In one embodiment, the bisulfite forsaid bisulfite-treated template was an aqueous solution of a bisulfitesalt (e.g. sodium bisulfite, magnesium bisulfite, etc.).

The present invention contemplates still other embodiments where treatedtemplate is employed in the context of amplifying on a solid support. Inone embodiment, the present invention contemplates a method ofamplifying nucleic acid on a solid support, comprising: a) providing apopulation of a known number of beads, and a population of a knownnumber of nucleic acid template molecules treated with bisulfite,wherein said known number of nucleic acid template molecules is lessthan the known number of beads; b) contacting said population of beadswith said population of nucleic acid template molecules under conditionssuch that at least a portion of said nucleic acid is non-covalentlyattached so as to create loaded beads comprising immobilized template;and c) amplifying at least a portion of said immobilized template so asto create immobilized amplified nucleic acid. In one embodiment, themethod further comprises: d) treating said immobilized amplified nucleicacid so as to release at least a portion from said loaded beads so as tocreate free amplified nucleic acid. In one embodiment, the methodfurther comprises: d) transferring at least a portion of saidimmobilized amplified nucleic acid to a non-bead solid support. In oneembodiment, the method further comprises: d) detecting at least aportion of said immobilized amplified nucleic acid. In one embodiment,the method further comprises: d) determining at least a portion of thesequence of the immobilized amplified nucleic acid on one or more beads.In one embodiment, determining at least a portion of the sequencecomprises use of nucleic acid hybridization probes, single baseextension, DNA sequencing and mass spectrometry (or other assay). In oneembodiment, the method further comprises: d) transcribing andtranslating the immobilized amplified nucleic acid. In one embodiment,each bead of step (a) comprises a forward and a reverse PCR primer.While it is not intended that the present invention be limited toparticular chemistries, in one embodiment, prior to step (a) saidforward and reverse PCR primers comprised 5′ amine modifications andwere attached to agarose beads comprising a plurality of primary aminereactive functional groups. It is not intended that the presentinvention be limited to the nature of the primers. In one embodiment,the primers have a region of complementarity to a gene associated withmethylation and/or methylation differences associated with disease. Inone embodiment, said forward and reverse PCR primers have a region thatis completely complementary to a portion of the vimentin gene. Inanother embodiment, said forward and reverse PCR primers have a regionthat is completely complementary to a portion of the RASF2A gene. In oneembodiment, said forward primer comprises a portion encoding anN-terminal epitope tag and said reverse primer comprises a portionencoding a C-terminal epitope tag. In one embodiment, the bisulfite forsaid bisulfite-treated template was an aqueous solution of a bisulfitesalt (e.g. sodium bisulfite, magnesium bisulfite, etc.).

In yet another embodiment employing treated template, the presentinvention contemplates a method of amplifying nucleic acid on a solidsupport, comprising: a) providing a population of a known number ofbeads, each bead comprising forward and reverse PCR primers linked tothe bead through a photocleavable linker, a population of a known numberof nucleic acid template molecules treated with bisulfite, wherein saidknown number of nucleic acid template molecules is less than the knownnumber of beads; and b) contacting said population of beads with saidpopulation of nucleic acid template molecules under conditions such thatat least a portion of said nucleic acid is amplified to create loadedbeads comprising immobilized amplified nucleic acid linked to the beadsthrough a photocleavable linker. In one embodiment, the method furthercomprises: c) exposing at least a portion of said immobilized amplifiednucleic acid to light so as to create free amplified nucleic acid. Inone embodiment, the method further comprises: c) exposing at least aportion of said immobilized amplified nucleic acid to light so as totransfer at least a portion of said immobilized amplified nucleic acidto a non-bead solid support. In one embodiment, the method furthercomprises: c) treating said immobilized amplified nucleic acid so as torelease at least a portion from said loaded beads so as to create freeamplified nucleic acid. In one embodiment, the method further comprises:c) transferring at least a portion of said immobilized amplified nucleicacid to a non-bead solid support. In one embodiment, the method furthercomprises: c) detecting at least a portion of said immobilized amplifiednucleic acid. In one embodiment, the method further comprises: c)determining at least a portion of the sequence of the immobilizedamplified nucleic acid on one or more beads. In one embodiment,determining at least a portion of the sequence comprises use of nucleicacid hybridization probes, single base extension, DNA sequencing andmass spectrometry (or other assay). In one embodiment, the methodfurther comprises: c) transcribing and translating the immobilizedamplified nucleic acid. While it is not intended that the presentinvention be limited to particular chemistries, in one embodiment, priorto step (a) said forward and reverse PCR primers comprised 5′ aminemodifications and were attached to agarose beads comprising a pluralityof primary amine reactive functional groups. It is not intended that thepresent invention be limited to the nature of the primers. In oneembodiment, the primers have a region of complementarity to a geneassociated with methylation and/or methylation differences associatedwith disease. In one embodiment, said forward and reverse PCR primershave a region that is completely complementary to a portion of thevimentin gene. In another embodiment, said forward and reverse PCRprimers have a region that is completely complementary to a portion ofthe RASF2A gene. In one embodiment, said forward primer comprises aportion encoding an N-terminal epitope tag and said reverse primercomprises a portion encoding a C-terminal epitope tag.

In some embodiments, it is desirable to control the amplification oftemplate on a solid support. For example, where it is desired that theamplification product on a bead (or other particle or nanoparticle) behomogeneous (or at least substantially homogeneous), it is useful tolimit the concentration of template such that the ratio of beads totemplate results in between 0 and 10 template molecules, more preferablybetween 1 and 5 template molecules, hybridizing to the primers. This isparticularly useful where multiplexing is desired (i.e. the simultaneousamplification of different templates). For multiplexing, the beads (orother particle or nanoparticle) can initially be treated separately, butthereafter mixed for simultaneous amplification. For example, in oneembodiment, first and second beads are mixed (after they were initiallytreated with first and second templates in the manner described herein)and thereafter amplified under conditions such that the amplifiedproduct on said first bead comprises greater than 90% first template,and the amplifed product on said second bead comprises greater than 90%second template. Unlike the prior art, such mixing of first and secondbeads can be done under non-emulsion conditions. In one embodiment, thepresent invention contemplates a method comprising a) providing templatein a primer-free solution and beads, said beads comprising covalentlyattached forward and reverse PCR primers, b) mixing said beads andtemplate under conditions such that said primers are extended, so as tocreate covalently attached extended products, c) washing said beadsunder conditions such that they are substantially free of template, andc) thermally cycling said beads such that said extended products areamplified. In one embodiment, the present invention contemplates amethod of amplifying nucleic acid on a solid support, comprising: a)providing i) a population of beads (or other particle or nanoparticle),each bead comprising one or more amplification primers, ii) a solutionof amplification reagents comprising a thermostable polymerase (butpreferably primer-free), and iii) a population of nucleic acid templatemolecules (again, preferably primer-free), b) mixing said beads and saidtemplate molecules in a first aliquot of said solution of amplificationreagents so as to create a mixture; c) treating the mixture underconditions such that at least a portion of said template moleculesnon-covalently bind to at least a portion of said beads to create boundtemplate, and at least a portion of said primers on at least a portionof said beads are extended by said polymerase, so as to create treatedbeads; d) manipulating said treated beads so as to remove at least aportion of said bound template so as to create manipulated beads; and e)contacting said manipulated beads with a second aliquot of said solutionof amplification reagents under conditions such that at least a portionof said extended primers is amplified to create loaded beads comprisingimmobilized amplified nucleic acid and unloaded beads lacking amplifiednucleic acid. In one embodiment, said manipulating of step d) compriseswashing said treated beads with a denaturing solution (e.g. a solutioncomprising NaOH). In one embodiment, prior to step d) between 1 and 10primers per bead are extended. In one embodiment, prior to step d) somebeads comprise no extended primers. In one embodiment, at step a) aknown concentration of beads is provided. In one embodiment, at step a)a known concentration of nucleic acid template molecules is provided. Inone embodiment, at step b) the number of template molecules to beads isless than one. In one embodiment, at step c) fewer than 50% of the beadscomprise non-covalently bound template. In one embodiment, theamplification primers comprise a sequence which provides a code. In oneembodiment, said code identifies the origin of the nucleic acidtemplates. In one embodiment, the origin of the nucleic acid template isa patient and the code identifies the patient. In one embodiment, saidcode identifies the bead. In one embodiment, each bead of step (a)comprises a forward and a reverse PCR primer.

In some embodiments, it is useful to have conditions that create loadedbeads comprising immobilized amplified nucleic acid and unloaded beadslacking amplified in order to control for homogeneity of the amplifiedproduct. In one embodiment, the present invention contemplates a methodof amplifying nucleic acid on a solid support, comprising: a) providingi) a population of beads (or other particle or nanoparticle), each beadcomprising forward and reverse PCR primers primers, ii) a solution ofamplification reagents comprising a thermostable polymerase, and iii) apopulation of nucleic acid template molecules, b) mixing said beads andsaid template molecules in a first aliquot of said solution ofamplification reagents so as to create a mixture; c) treating themixture under conditions such that at least a portion of said templatemolecules non-covalently bind to at least a portion of said beads tocreate bound template, and at least a portion of said primers on atleast a portion of said beads are extended by said polymerase, so as tocreate treated beads; d) washing said treated beads with a denaturingsolution so as to create manipulated beads; and e) contacting saidmanipulated beads with a second aliquot of said solution ofamplification reagents under conditions such that at least a portion ofsaid extended primers is amplified to create loaded beads comprisingimmobilized amplified nucleic acid and unloaded beads lacking amplifiednucleic acid. In one embodiment, the method further comprises: (f)treating said immobilized amplified nucleic acid so as to release atleast a portion from said loaded beads so as to create free amplifiednucleic acid. In another embodiment, the method further comprises: (f)transferring at least a portion of said immobilized amplified nucleicacid to a non-bead solid support. In one embodiment, prior to step (a)said forward and reverse PCR primers comprised 5′ amine modificationsand were attached to agarose beads comprising a plurality of primaryamine reactive functional groups. In one embodiment, said forward andreverse PCR primers have a region that is completely complementary to aportion of disease-related gene (e.g. the APC gene segment 3). In oneembodiment, said forward primer comprises a portion encoding anN-terminal epitope tag and said reverse primer comprises a portionencoding a C-terminal epitope tag. In one embodiment, said mixture iscreated under the conditions such that the ratio of the number ofnucleic acid template molecules to the number of beads is between 0.1:1and 2:1. In one embodiment, said mixture is created under the conditionssuch that the ratio of the number of nucleic acid template molecules tothe number of beads is between 2:1 and 500,000:1. In one embodiment, theratio of the number of nucleic acid template molecules to the number ofbeads is between 1000:1 and 100,000:1. In one embodiment, the ratio ofthe number of nucleic acid template molecules to the number of beads isbetween 10,000:1 and 100,000:1. In one embodiment, the ratio of thenumber of nucleic acid template molecules to the number of beads isbetween 1000:1 and 10,000:1. In one embodiment, the percentage ofunloaded beads is between approximately 50% and 95%, as measured byfluorescence. In one embodiment, the percentage of loaded beads isbetween approximately 1% and 5%, as measured by fluorescence. In oneembodiment, the percentage of loaded beads is between approximately 5%and 50%, as measured by fluorescence (an assay for which is describedbelow).

The present invention contemplates other embodiments of the method forcreating loaded and unloaded beads (or particles or nanoparticles). Inone embodiment, the present invention contemplates a method amplifyingnucleic acid on a solid support, comprising: a) providing i) apopulation of a known concentration of beads (or particles ornanoparticles), each bead comprising one or more amplification primers,ii) a solution of amplification reagents comprising a thermostablepolymerase, and iii) a population of a known concentration of nucleicacid template molecules; b) mixing said beads and said templatemolecules in a first aliquot of said solution of amplification reagentsso as to create a mixture under the conditions such that the ratio ofthe number of nucleic acid template molecules to the number of beads isbetween 1:1 and 10,000:1; c) treating the mixture under conditions suchthat at least a portion of said template molecules non-covalently bindto at least a portion of said beads to create bound template, and atleast a portion of said primers on at least a portion of said beads areextended by said polymerase, so as to create treated beads; d)manipulating said treated beads so as to remove at least a portion ofsaid bound template so as to create manipulated beads; and e) contactingsaid manipulated beads with a second aliquot of said solution ofamplification reagents under conditions such that at least a portion ofsaid extended primers is amplified to create loaded beads comprisingimmobilized amplified nucleic acid and unloaded beads lacking amplifiednucleic acid. In one embodiment, the method further comprises: (f)treating said immobilized amplified nucleic acid so as to release atleast a portion from said loaded beads so as to create free amplifiednucleic acid. In another embodiment, the method further comprises: (f)transferring at least a portion of said immobilized amplified nucleicacid to a non-bead solid support. In one embodiment, each bead of step(a) comprises a forward and a reverse PCR primer. In one embodiment,said manipulating comprises washing said treated beads with a denaturingsolution (e.g. a solution comprising NaOH). In one embodiment, saidwashing removes the majority of said non-covalently bound template. Inone embodiment, prior to step (a) said forward and reverse PCR primerscomprised 5′ amine modifications and were attached to agarose beadscomprising a plurality of primary amine reactive functional groups. Inone embodiment, said forward and reverse PCR primers have a region thatis completely complementary to a portion of disease-related gene (e.g.the APC gene segment 3). In one embodiment, said forward primercomprises a portion encoding an N-terminal epitope tag and said reverseprimer comprises a portion encoding a C-terminal epitope tag. In oneembodiment, the percentage of unloaded beads is between approximately50% and 95%, as measured by fluorescence. In one embodiment, thepercentage of loaded beads is between approximately 1% and 5%, asmeasured by fluorescence. In one embodiment, the percentage of loadedbeads is between approximately 5% and 50%, as measured by fluorescence(an assay for which is described below). In one embodiment, the ratio ofthe number of nucleic acid template molecules to the number of beads isbetween 1:1 and 10:1. In one embodiment, the ratio of the number ofnucleic acid template molecules to the number of beads is between 10:1and 100:1. In one embodiment, the ratio of the number of nucleic acidtemplate molecules to the number of beads is between 100:1 and 1,000:1.

Still other embodiments of methods creating loaded and unloaded beads(or other particle or nanoparticle) employ lower ratios. In oneembodiment, the present invention contemplates A method of amplifyingnucleic acid on a solid support, comprising: a) providing i) apopulation of a known concentration of beads, each bead comprising oneor more amplification primers, ii) a solution of amplification reagentscomprising a thermostable polymerase, and iii) a population of a knownconcentration of nucleic acid template molecules, b) mixing said beadsand said template molecules in a first aliquot of said solution ofamplification reagents so as to create a mixture under the conditionssuch that the ratio of the number of nucleic acid template molecules tothe number of beads is between 0.1:1 and 2:1; c) treating the mixtureunder conditions such that at least a portion of said template moleculesnon-covalently bind to at least a portion of said beads to create boundtemplate, and at least a portion of said primers on at least a portionof said beads are extended by said polymerase, so as to create treatedbeads; d) exposing said treated beads to a denaturing solution so as tocreate manipulated beads; and e) contacting said manipulated beads witha second aliquot of said solution of amplification reagents underconditions such that at least a portion of said extended primers isamplified to create loaded beads comprising immobilized amplifiednucleic acid and unloaded beads lacking amplified nucleic acid. In oneembodiment, the method further comprises: (f) treating said immobilizedamplified nucleic acid so as to release at least a portion from saidloaded beads so as to create free amplified nucleic acid. In anotherembodiment, the method further comprises: (f) transferring at least aportion of said immobilized amplified nucleic acid to a non-bead solidsupport. In one embodiment, each bead of step (a) comprises a forwardand a reverse PCR primer. In one embodiment, said denaturing solutioncomprises NaOH and said exposing comprises at least two washings of thebeads. In one embodiment, said washings remove at least a portion ofsaid non-covalently bound template. In one embodiment, said washingsremoves the majority of said non-covalently bound template. In oneembodiment, the percentage of unloaded beads is between approximately50% and 99%, as measured by fluorescence. In one embodiment, thepercentage of loaded beads is between approximately 0.1% and 2%, asmeasured by fluorescence (an assay for which is described below).

In one embodiment, the present invention contemplates generating andcapturing nascent proteins (or portions thereof) or peptides on the samesolid support. In one embodiment, the present invention contemplates asurface comprising captured nascent protein, or fragment thereof, andamplified product encoding said nascent protein or fragment thereof (andmethods of making such a surface). In one embodiment, the presentinvention contemplates a method of generating and capturing nascentproteins (or portions thereof) and peptides, comprising: a) providingnucleic acid encoding a protein (or portions thereof), and a pluralityof beads (or other particle or nanoparticle), each bead comprising oneor more amplification primers; b) contacting said beads with saidnucleic acid under conditions such that at least a portion of saidnucleic acid is amplified to create treated beads comprising immobilizedamplified nucleic acid; and c) producing nascent protein from at least aportion of said immobilized amplified nucleic acid on said treated beadsby (preferably cell free) expression to create expressed beads, whereinat least a portion of said nascent protein (or portion thereof) iscaptured on said expressed beads. It is not intended that the presentinvention be limited by the manner in which said protein (or portionthereof) is captured. In one embodiment, each of said beads, prior tostep c), comprises a plurality of first binding agents on the beadsurface, said first binding agents capable of binding said nascentprotein. In one embodiment, said first binding agents comprise chemicalmoieties.

It is not intended that the present invention be limited to particularchemical moieties; a variety are contemplated. For example, in oneembodiment, said chemical moieties are selected from the groupconsisting of amines, sulfhydryls, carboxyls, epoxy, and aldehydemoieties.

In one embodiment, said binding agents are ligands. For example, in oneembodiment, said first binding agents are selected from the groupconsisting of antibodies, aptamers, streptavidin and avidin. It is notintended that the present invention be limited to only one bindingagent. For example, in one embodiment, each of said beads, prior to stepc), comprises a plurality of first binding agents on the bead surface,said first binding agents capable of binding a plurality of secondbinding agents, said second binding agents capable of binding saidnascent protein. In yet another embodiment, each of said beads, prior tostep c), comprises a plurality of first binding agents on the beadsurface, said first binding agents capable of binding a plurality ofsecond binding agents, said second binding agents capable of binding aplurality of third binding agents, said third binding agents capable ofbinding said nascent protein. In one embodiment, said first bindingagent comprises biotin, said second binding agent comprises avidin, andsaid third binding agent comprises biotinylated antibody. In the latterembodiment, it is preferred that said avidin is a tetramer.

While not intending to limit the invention to any particular mechanism,the present invention contemplates, in one embodiment, that saidcontacting of step b) results in at least a portion of said nucleic acidannealing to said one or more amplification primers. Moreover, in oneembodiment, after said annealing at least a portion of said primers areextended (e.g. wherein said conditions comprise use of a polymerase). Itis preferred that after said primers are extended, the beads are treatedunder denaturing conditions.

Once the protein (or portions thereof) or peptide is captured, the beadcan be used in a variety of assays. For example, in one embodiment, themethod (above) further comprises, after step c), sequencing at least aportion of said nascent protein. In another embodiment, the method(above) further comprises after step c), determining whether saidnascent protein comprises truncated protein (which can be done by ELISAusing antibodies, mass spec, etc.).

Once the protein (or portions thereof) or peptide is captured, it can betransferred to another solid support, including but not limited tonon-bead solid supports. For example, in one embodiment, the method(above) further comprises, after step c), transferring at least aportion of said nascent protein to a non-bead solid support, so as tocreate transferred nascent protein. In some embodiments, the assaying ofthe protein (or portions thereof) is done after such a transfer. Forexample, in one embodiment, the method (above) further comprises thestep of sequencing at least as portion of said transferred nascentprotein (or portion thereof) or peptide.

It is not intended that the present invention be limited to onlytransferring captured protein. In one embodiment, the method furthercomprises, after step c), transferring at least a portion of saidnascent protein and at least a portion of said amplified nucleic acid toa non-bead solid support, so as to create transferred nascent proteinand transferred nucleic acid. In some embodiments, the assaying of thenucleic acid (or portions thereof is done after such a transfer. Forexample, in one embodiment, the method further comprises the step ofsequencing at least a portion of said transferred nucleic acid.

In one embodiment, the present invention contemplates, as a compositionof matter, “loaded beads,” i.e. beads (or other particle ornanoparticle) with captured protein(s) or peptide(s). In one embodiment,the “loaded beads” further comprises nucleic acid encoding said capturedprotein(s) or peptide(s). It is not intended that the present inventionbe limited by the methods by which this is achieved. Nonetheless, anillustrative embodiment is a method of generating and capturing nascentproteins, comprising: a) providing i) nucleic acid encoding a protein orfragment thereof, ii) a plurality of beads (or other particle ornanoparticle), each bead comprising one or more amplification primersand one or more first binding molecules, and iii) a population of secondbinding molecules capable of binding to said protein and said firstbinding molecules; b) contacting said beads with said nucleic acid underconditions such that at least a portion of said nucleic acid isamplified to create treated beads comprising immobilized amplifiednucleic acid; c) contacting said treated beads with said second bindingmolecules under conditions such that at least a portion of said firstbinding molecules bind to at least a portion of said second bindingmolecules so as to create capture beads; and d) producing nascentprotein or fragments thereof from at least a portion of said immobilizedamplified nucleic acid on said capture beads by cell free expression, atleast a portion of said nascent protein or fragments thereof interactingwith at least a portion of said second binding molecules so as togenerate loaded beads comprising captured nascent protein or capturedfragments thereof. In one embodiment, the present invention contemplatesthe loaded beads generated according to this method as a composition ofmatter.

As mentioned above, it is not intended that the present invention belimited to particular chemical moieties; a variety are contemplated. Forexample, in one embodiment, said chemical moieties are selected from thegroup consisting of amines, sulfhydryls, carboxyls, epoxy, and aldehydemoieties.

In one embodiment, said binding agents are ligands. For example, in oneembodiment, the present invention contemplates that said first bindingagents comprise biotin. In another embodiment, the present inventioncontemplates that said second binding agents comprise antibody havingaffinity for said nascent protein or fragments thereof.

As with other embodiments discussed above, the protein or fragmentthereof can be assayed after capture, either before or after transfer(e.g. transfer to another solid support, including non-bead supports).For example, in one embodiment, the method further comprises e)sequencing at least a portion of said nascent protein. In anotherembodiment, the method further comprises e) transferring at least aportion of said captured nascent protein to a non-bead solid support, soas to create transferred nascent protein, and thereafter f) sequencingat least a portion of said transferred nascent protein (or fragmentthereof).

As with other embodiments discussed above, more than just the protein(or fragment) can be transferred. In one embodiment, the method furthercomprises e) transferring at least a portion of said captured nascentprotein and at least a portion of said amplified nucleic acid, so as tocreate transferred nascent protein and transferred nucleic acid. In oneembodiment, the method further comprises f) sequencing at least aportion of said transferred nucleic acid.

As with other embodiments discussed above, it is not intended that thepresent invention be limited by the nature of the assay. In oneembodiment, the method further comprises after step d) determiningwhether said nascent protein comprises truncated protein. Suchdetermining can be done by a variety of methods, including sequencing,ELISA (with antibodies to the C-terminus), or mass spec (in order todetect a smaller peptide).

In one embodiment, the method further comprises: (e) treating saidcaptured nascent protein so as to release at least a portion from saidloaded beads so as to create free nascent protein. It is not intendedthat the present invention be limited by the configuration which permitstransfer. In a preferred embodiment, transfer is photo-transfer. In oneembodiment, biotin (or another ligand) is linked to said beads via aphotocleavable linker and the treating of step e) comprises exposingsaid photocleavable linker to light.

In one embodiment, each bead (or other particle or nanoparticle) of step(a) comprises a forward and a reverse PCR primer. It is not intendedthat the present invention be limited by the chemistry by which theprimers are attached. In one embodiment, prior to step (a) said forwardand reverse PCR primers comprised 5′ amine modifications and wereattached to beads (e.g. agarose beads) comprising a plurality of primaryamine reactive functional groups.

The present invention contemplates other embodiments for generating“loaded beads.” For example, in one embodiment, the present inventioncontemplates a method of generating and capturing nascent proteins (orfragments thereof), comprising: a) providing nucleic acid encoding aprotein or fragment thereof, a plurality of beads (or other particle ornanoparticle), each bead comprising one or more amplification primersand one or more first binding molecules, a population of second bindingmolecules capable of binding to said first binding molecules, and apopulation of third binding molecules capable of binding to said secondbinding molecules and said protein or fragment thereof; b) contactingsaid beads with said nucleic acid under conditions such that at least aportion of said nucleic acid is amplified to create treated beadscomprising immobilized amplified nucleic acid; c) contacting saidtreated beads with said second binding molecules under conditions suchthat at least a portion of said first binding molecules bind to at leasta portion of said second binding molecules so as to create conjugatedbeads; d) contacting said conjugated beads with said third bindingmolecules under conditions such that at least a portion of said secondbinding molecules bind to at least a portion of said third bindingmolecules so as to create capture beads; and e) producing nascentprotein or fragment thereof from at least a portion of said immobilizedamplified nucleic acid on said capture beads by cell free expression, atleast a portion of said nascent protein or fragment thereof interactingwith at least a portion of said third binding molecules so as togenerate loaded beads comprising captured nascent protein or capturedfragment thereof.

As with other embodiments discussed above, the protein or fragmentthereof can be assayed after capture, either before or after transfer(e.g. transfer to another solid support, including non-bead supports).In one embodiment, the method further comprises f) sequencing at least aportion of said nascent protein. In another embodiment, the methodfurther comprises f) transferring at least a portion of said capturednascent protein to a non-bead solid support, so as to create transferrednascent protein and g) sequencing at least a portion of said transferrednascent protein.

As with other embodiments discussed above, more than just the protein(or fragment) can be transferred. For example, in one embodiment, themethod further comprises f) transferring at least a portion of saidcaptured nascent protein and at least a portion of said amplifiednucleic acid, so as to create transferred nascent protein andtransferred nucleic acid. In one embodiment, after transferring, themethod further comprises g) sequencing at least a portion of saidtransferred nucleic acid.

As with other embodiments discussed above, it is not intended that thepresent invention be limited by the nature of the assay. In oneembodiment, the method further comprises after step e) determiningwhether said nascent protein comprises truncated protein. Suchdetermining can be done by a variety of methods, including sequencing,ELISA (with antibodies to the C-terminus), or mass spec (in order todetect a smaller peptide).

In one embodiment, the binding molecules are ligands. For example, inone embodiment said first binding molecules comprise biotin, said secondbinding molecules comprise streptavidin and said third binding moleculescomprise biotinylated antibody, said antibody having affinity for saidnascent protein or fragment thereof.

In one embodiment, the method further comprises: (f) treating saidcaptured nascent protein or fragment thereof so as to release at least aportion from said loaded beads so as to create free nascent protein orfree fragment thereof. It is not intended that the present invention belimited to the method of transfer. In one embodiment of phototransfer,said biotinylated antibody comprises biotin linked via a photocleavablelinker to said antibody and said treating of step f) comprises exposingsaid photocleavable linker to light.

In one embodiment, each bead of step (a) comprises a forward and areverse PCR primer. The attachment can be done in a variety of ways. Inone embodiment, prior to step (a) said forward and reverse PCR primerscomprised 5′ amine modifications and were attached to agarose beadscomprising a plurality of primary amine reactive functional groups.

In yet another embodiment of creating “loaded beads,” the presentinvention contemplates a method of generating and capturing truncatedprotein, comprising: a) providing nucleic acid encoding a truncatedprotein, a plurality of beads (or other particle or nanoparticle), eachbead comprising one or more amplification primers and one or more firstbinding molecules, a population of second binding molecules capable ofbinding to said first binding molecules, and a population of thirdbinding molecules capable of binding to said second binding moleculesand capturing said truncated protein; b) contacting said beads with saidnucleic acid under conditions such that at least a portion of saidnucleic acid is amplified to create treated beads comprising immobilizedamplified nucleic acid; c) contacting said treated beads with saidsecond binding molecules under conditions such that at least a portionof said first binding molecules bind to at least a portion of saidsecond binding molecules so as to create conjugated beads; d) contactingsaid conjugated beads with said third binding molecules under conditionssuch that at least a portion of said second binding molecules bind to atleast a portion of said third binding molecules so as to create capturebeads; and e) producing truncated protein from at least a portion ofsaid immobilized amplified nucleic acid on said capture beads by cellfree expression, said third binding molecules capturing at least aportion of said truncated protein so as to generate loaded beadscomprising captured truncated protein.

As with other embodiments discussed above, the protein or fragmentthereof can be assayed after capture, either before or after transfer(e.g. transfer to another solid support, including non-bead supports).In one embodiment, the method further comprises f) sequencing at least aportion of said nascent protein. In another embodiment, the methodfurther comprises f) transferring at least a portion of said capturednascent protein to a non-bead solid support, so as to create transferrednascent protein and g) sequencing at least a portion of said transferrednascent protein.

As with other embodiments discussed above, more than just the protein(or fragment) can be transferred. For example, in one embodiment, themethod further comprises f) transferring at least a portion of saidcaptured nascent protein and at least a portion of said amplifiednucleic acid, so as to create transferred nascent protein andtransferred nucleic acid. When nucleic acid is transferred, it can alsobe assayed. In one embodiment, the method further comprises g)sequencing at least a portion of said transferred nucleic acid.

In one embodiment, the method further comprises after step e)determining whether said nascent protein comprises truncated protein. Aswith other embodiments discussed above, it is not intended that thepresent invention be limited by the nature of the assay (e.g. gelelectrophoresis, ELISA with antibodies to the C-terminus, mass spec,etc.).

As with other embodiments, the binding molecules may be ligands. In oneembodiment, said first binding molecules comprise biotin, said secondbinding molecules comprise streptavidin and said third binding moleculescomprise biotinylated antibody.

In one embodiment, the method further comprising: (f) treating saidcaptured truncated protein so as to release at least a portion from saidloaded beads so as to create free truncated protein. As with otherembodiments, it is not intended that the invention be limited to anyparticular transfer mechanism. Nonetheless, a preferred transfer isphototransfer. In one embodiment, said biotinylated antibody comprisesbiotin linked via a photocleavable linker to said antibody. Thus, in oneembodiment, the method comprises exposing said photocleavable linker tolight.

In one embodiment, each bead of step (a) comprises a forward primerencoding a first epitope and a reverse PCR primer encoding a secondepitope. Again, it is not intended that the invention be limited by theattachment chemistry. Nonetheless, in one embodiment, prior to step (a)said forward and reverse PCR primers comprised 5′ amine modificationsand were attached to agarose beads comprising a plurality of primaryamine reactive functional groups.

In all of the above-discussed embodiments, the protein, protein portion,protein fragment, truncated protein or peptide can be encoded by avariety of disease related genes or portions of such genes. In oneembodiment, at least a portion of said truncated protein is encoded by aportion of the APC gene.

In one embodiment, a plurality of biomolecule species are produced,sorted on beads, in multiplexed fashion, i.e. using one or a fewreactions, each within a single reactor. It is not intended that thepresent invention be limited by the nature of the biomolecules. In oneembodiment, the biomolecules are peptides or proteins. In oneembodiment, the biomolecules are nucleic acids, nucleosides, nucleotidesor polymers thereof which are useful directly or used to subsequentlydirect de novo protein synthesis, hence producing, in multiplexedfashion, a plurality protein or peptide biomolecules also sorted on thesame beads.

The produced biomolecules on beads can be used as probes, targets oranalytes, for example, for various parallel, massively parallel ormultiplexed analyses such as DNA sequencing and/or mutation detection aswell as various genome-wide and proteome-wide analyses.

However, the plurality of produced biomolecules can be for a variety ofuses. These biomolecules are not intended to be limited to any one use,and henceforth will be referred to “features”, as is commonly used inthe art of microarrays. Compared to conventional approaches, it is farmore desirable to utilize truly multiplexed methods to produce saidfeatures on beads, with one or a few reactions, each within a singlereactor for example.

In one preferred embodiment, a method is disclosed for the multiplexedproduction of a plurality of DNA features, sorted on beads, usingsolid-phase bridge PCR amplification with a given amplification primerset on each bead species. Each bead can amplify a plurality template DNAmolecules or more preferably, each bead can clone (amplify) a singletemplate molecule, all performed with the entire bead population in asingle reactor. In either case, the DNA amplicon on the beads cansubsequently be used for multiplexed cell-free (in vitro) proteinsynthesis on the beads, producing a bead-sorted library of in vitroexpressed proteins (BS-LIVE-PRO). This is achieved by cell-free (invitro) transcription and translation of the entire bead population andcapturing molecules of each produced protein species on the sameDNA-encoded bead from which they were made, all in a single reactor(e.g. in a single tube).

The single-molecule solid-phase bridge PCR amplification approach isparticularly useful, since in that embodiment a single primer set (pair)on a population of beads can be used to amplify a plurality of differentDNA templates in a single reactor (e.g. amplify different cDNAs from acDNA library pertaining to different gene coding sequences), yet thedifferent amplicon species, arising from the single template molecules,remain sorted on different beads (for example, all template moleculeshaving some common sequences, e.g. on 5′ and 3′ ends of the templateDNA). This can be useful in the highly multiplexed manufacturing ofprobe or target type features for beads, e.g. to produce proteomelibraries sorted on beads. However, the method is also useful in theproduction of analyte type “features” for diagnostic applications, forexample, where a population of molecules (e.g. population of moleculescorresponding to a particular gene or fragment thereof) needs to bequeried for sub-populations (e.g. a minor sub-population of those genemolecules containing a disease causing mutation).

Furthermore, in one embodiment, the solid-phase bridge PCR usesamplification primers attached to the beads, with no soluble primers(i.e. it is preferred that free primers are not added), and hence thePCR amplicon is also restricted to the beads. This facilitates fullmultiplexing, should the need exist to target single molecules of aplurality of different DNA template species, using different specificprimer sets. For example, with this approach, it is possible to clone(amplify) single template molecules on beads, whereby the differentprimer sets on different beads target single template moleculescorresponding to different fragments of a gene, all performed with theentire bead and template population within a single reactor (using oneor a few reactions).

Methods are also disclosed that pertain to the photo-transfer offeatures, produced as described above or in any other manner, from beadsto planar substrates (or to wells into which the beads fit), such asmicroarray substrates, in order to create microarray features from saidbeads.

More broadly however, this invention also relates to methods andcompositions for the photo-transfer of substances and compounds from onesurface to another, without restrictions on the types or numbers ofsubstances and compounds, and without restrictions on the typessurfaces, and is therefore also applicable to a much wider range offields.

In a preferred embodiment, compounds such as macromolecules orsubstances such as nanoparticles (particles of between 1 and 100 nm indiameter) or cells, which serve as probes, analytes or targets forexample, are attached to a surface through photocleavable linkers, saidsurface allowed to contact a second surface and then said substances orcompounds photo-released under conditions to allow transfer to saidsecond surface. Surfaces can be, but are not limited to, beads, glassslides, metallic surfaces, plastic or polymeric surfaces and othersurfaces used for biomolecular detection. In some embodiments, it is notstrictly necessary that said photo-transferred compounds or substancesbe in physical contact with said second surface, but in close proximityinstead.

In one embodiment, the method also involves the photo-transfer of acoding agent. For example, in one preferred embodiment, the presentinvention contemplates a method for co-transferring coding agents andcompounds or substances from a bead to a surface and using said codingagents to determine the identity of said co-transferred compound orsubstance or the identity of said bead.

In some instances, it is desired to photo-transfer more than just onecompound or substance. For example, in some embodiments, any combinationof various substances and/or compounds are photo-transferred.

The present invention also contemplates sorting out and/or enrichingsubpopulations of biomolecules on solid supports, including enrichingsubpopulations of beads containing biomolecules. In one embodiment, thepresent invention contemplates method of enriching a subpopulation ofbeads in a mixture, comprising: a) providing a mixture comprising i) aplurality of first beads, said first beads comprising immobilized firstamplified product, said first amplified product encoding a first nascentprotein or fragment thereof, and ii) a plurality of second beads, saidsecond beads comprising immobilized second amplified product, saidsecond amplified product encoding a second nascent protein or fragmentthereof; b) exposing said mixture to translation system under conditionssuch that said first and second nascent proteins or fragments thereofare generated from at least a portion of said first and secondimmobilized amplified products, c) capturing at least a portion of saidfirst nascent protein or fragment thereof on said first bead andcapturing at least a portion of said second nascent protein or fragmentthereof on said second bead, so as to create a mixture of beadscomprising captured proteins or fragments thereof; and d) contactingsaid mixture of beads comprising captured proteins or fragments thereofwith a ligand with affinity for said first nascent protein or fragmentthereof, said contacting performed under conditions such that at least aportion of said first beads are separated from said mixture, therebyenriching a subpopulation of beads. In one embodiment, each of saidbeads, prior to step c), comprises a plurality of reactive chemicalmoieties on the bead surface, said moieties capable of reacting withsaid nascent protein. In one embodiment, said ligand comprises anantibody. In one embodiment, said antibody is attached to magneticbeads. In one embodiment, said conditions of step d) comprise exposureof said mixture to a magnet. In one embodiment, said exposure to amagnet creates precipitated beads and a supernatant. In one embodiment,said conditions of step d) further comprise removing said supernatant orsubstantially all (e.g. 90% or more) of said supernatant, so as tocreate an isolated precipitate. In one embodiment, said conditions ofstep d) further comprise removing said precipitated beads orsubstantially all (e.g. 90% or more) of said precipitated beads, so asto create a depleted supernatant. In one embodiment, the ratio of firstbeads to second beads in said mixture of step a) is 50:50. In oneembodiment, said isolated precipitate is contaminated with less than 30%of second beads. In one embodiment, said isolated precipitate iscontaminated with less than 10% of second beads. In one embodiment, saidisolated precipitate is contaminated with less than 1% of second beads.In one embodiment, said mixture of step a) further comprises a pluralityof third beads. In one embodiment, said third beads lack immobilizedamplified product. In one embodiment, said third beads compriseimmobilized third amplified product, said third amplified productencoding a third nascent protein or fragment thereof. In one embodiment,said first immobilized amplified product comprises at least a portion ofa disease-related (including but not limited to cancer-related) gene. Inone embodiment, said disease-related gene is selected from the groupconsisting of the APC gene, the NF1 gene, the NF2 gene, the BRCA1 gene,the BRCA2 gene, the Kras gene, and the p53 gene. In one embodiment, thenumber of second beads in said mixture is less than the number of saidfirst beads.

It is not intended that the present invention be limited to the numberor nature of ligands employed to enrich or sort subpopulations. In oneembodiment, the present invention contemplates a method of enriching asubpopulation of beads (or other particle or nanoparticle) in a mixture,comprising: a) providing a mixture comprising i) a plurality of firstbeads, said first beads comprising immobilized first amplified product,said first amplified product encoding a first nascent protein orfragment thereof, and ii) a plurality of second beads, said second beadscomprising immobilized second amplified product, said second amplifiedproduct encoding a second nascent protein or fragment thereof; b)exposing said mixture to a translation system under conditions such thatsaid first and second nascent proteins or fragments thereof aregenerated from said first and second immobilized amplified products, c)capturing said first nascent protein or fragment thereof on said firstbead and capturing said second nascent protein or fragment thereof onsaid second bead, so as to create a mixture of beads comprising capturedproteins or fragments thereof; d) contacting said mixture of beadscomprising captured proteins or fragments thereof with a first ligandwith affinity for said first nascent protein or fragment thereof, so asto create a mixture of treated beads; e) contacting said mixture oftreated beads with a second ligand, said second ligand having affinityfor said first ligand, said contacting performed under conditions suchthat at least a portion of said first beads are separated from saidmixture, thereby enriching a subpopulation of beads. In one embodiment,each of said beads, prior to step c), comprises a plurality of reactivechemical moieties on the bead surface, said moieties capable of reactingwith said nascent protein. In one embodiment, said first ligandcomprises a first antibody. In one embodiment, said second ligandcomprises a second antibody. In one embodiment, said second antibody isattached to magnetic beads. In one embodiment, said conditions of stepe) comprise exposure of said mixture to a magnet. In one embodiment,said exposure to a magnet creates precipitated beads and a supernatant.In one embodiment, said conditions of step e) further comprise removingsaid supernatant or substantially all (90% or more) of said supernatant,so as to create an isolated precipitate. In one embodiment, saidconditions of step e) further comprise removing said precipitated beadsor substantially all (90% or more) of said precipitated beads, so as tocreate a depleted supernatant. In one embodiment, the ratio of firstbeads to second beads in said mixture of step a) is 50:50. In oneembodiment, said isolated precipitate is contaminated with less than 30%of second beads. In one embodiment, said isolated precipitate iscontaminated with less than 10% of second beads. In one embodiment, saidisolated precipitate is contaminated with less than 1% of second beads.In one embodiment, said mixture of step a) further comprises a pluralityof third beads. In one embodiment, said third beads lack immobilizedamplified product. In one embodiment, said third beads compriseimmobilized third amplified product, said third amplified productencoding a third nascent protein or fragment thereof. In one embodiment,said first immobilized amplified product comprises at least a portion ofa disease-related (including but not limited to cancer-related) gene. Inone embodiment, said disease-related gene is selected from the groupconsisting of the APC gene, the NF1 gene, the NF2 gene, the BRCA1 gene,the BRCA2 gene, the Kras gene, and the p53 gene. In one embodiment, thenumber of second beads in said mixture is less than the number of saidfirst beads.

In one embodiment, the present invention contemplates sorting out orenriching populations such that wild-type full-length protein isseparated from truncated protein. In one embodiment, the presentinvention contemplates a method of enriching a subpopulation of beads,comprising: a) providing a mixture comprising i) a plurality of firstbeads, said first beads comprising immobilized first amplified product,said first amplified product encoding a truncated version of a firstprotein, and ii) a plurality of second beads, said second beadscomprising immobilized second amplified product, said second amplifiedproduct encoding an untruncated version of said first protein, whereinthe number of first beads in said mixture is less than the number ofsaid second beads; b) exposing said mixture to a translation systemunder conditions such that said truncated and untruncated versions ofsaid first protein are generated from at least a portion of said firstand second immobilized amplified products, c) capturing said truncatedversion of said first protein on said first bead and capturing saiduntruncated version of said first protein on said second bead, so as tocreate a mixture of beads comprising captured proteins or truncatedfragments thereof; and d) contacting said mixture of beads comprisingcaptured proteins or truncated fragments thereof with a ligand withaffinity for said untruncated version of said first protein, so as tocreate a mixture of treated beads, said contacting performed underconditions such that at least a portion of said second beads areseparated from said mixture, thereby enriching a subpopulation of beadscomprising truncated protein. In one embodiment, each of said beads,prior to step c), comprises a plurality of reactive chemical moieties onthe bead surface, said moieties capable of reacting with and capturingsaid nascent protein. In one embodiment, said ligand comprises anantibody. In one embodiment, said antibody has affinity for a region ofsaid untruncated version of said first protein that is lacking in saidtruncated protein said antibody is attached to magnetic beads. In oneembodiment, said conditions of step d) comprise exposure of said mixtureto a magnet. In one embodiment, said exposure to a magnet createsprecipitated beads and a supernatant. In one embodiment, said conditionsof step d) further comprise removing said supernatant or substantiallyall (905 or more) of said supernatant, so as to create an isolatedprecipitate. In one embodiment, said conditions of step d) furthercomprise removing said precipitated beads or substantially all (90% ormore), so as to create a depleted supernatant. In one embodiment, theratio of first beads to second beads in said mixture of step a) is lessthan 1:10. In one embodiment, said isolated precipitate is contaminatedwith less than 5% of said first beads. In one embodiment, said isolatedprecipitate is contaminated with less than 2% of said first beads. Inone embodiment, said isolated precipitate is contaminated with less than1% of said first beads. In one embodiment, said mixture of step a)further comprises a plurality of third beads. In one embodiment, saidthird beads lack immobilized amplified product. In one embodiment, saidthird beads comprise immobilized third amplified product, said thirdamplified product encoding a third nascent protein or fragment thereof.In one embodiment, said first immobilized amplified product comprises atleast a portion of a disease-related (including but not limited tocancer-related) gene. In one embodiment, said disease-related gene isselected from the group consisting of the APC gene, the NF1 gene, theNF2 gene, the BRCA1 gene, the BRCA2 gene, the Kras gene, and the p53gene.

In yet another embodiment of sorting out and/or enriching for truncatedprotein, the present invention contemplates a method of enriching asubpopulation of beads, comprising: a) providing a mixture comprising i)a plurality of first beads, said first beads comprising immobilizedfirst amplified product, said first amplified product encoding atruncated version of a first protein, and ii) a plurality of secondbeads, said second beads comprising immobilized second amplifiedproduct, said second amplified product encoding an untruncated versionof said first protein, wherein the number of first beads in said mixtureis less than the number of said second beads; b) exposing said mixtureto a translation system under conditions such that said truncated anduntruncated versions of said first protein are generated from at least aportion of said first and second immobilized amplified products, c)capturing said truncated version of said first protein on said firstbead and capturing said untruncated version of said first protein onsaid second bead, so as to create a mixture of beads comprising capturedproteins or truncated fragments thereof; d) contacting said mixture ofbeads comprising captured proteins or truncated fragments thereof with afirst ligand with affinity for said untruncated version of said firstprotein, so as to create a mixture of treated beads; and e) contactingsaid mixture of treated beads with a second ligand, said second ligandhaving affinity for said first ligand, said contacting performed underconditions such that at least a portion of said first beads areseparated from said mixture, thereby enriching a subpopulation of beadscomprising truncated protein. In one embodiment, each of said beads,prior to step c), comprises a plurality of reactive chemical moieties onthe bead surface, said moieties capable of reacting with said nascentprotein. In one embodiment, said first ligand comprises a firstantibody. In one embodiment, said antibody has affinity for a region ofsaid untruncated version of said first protein that is lacking in saidtruncated protein. In one embodiment, said second ligand comprises asecond antibody. In one embodiment, said second antibody is attached tomagnetic beads. In one embodiment, said conditions of step e) compriseexposure of said mixture to a magnet. In one embodiment, said exposureto a magnet creates precipitated beads and a supernatant. In oneembodiment, said conditions of step e) further comprise removing saidsupernatant or substantially all (90% or more) of said supernatant, soas to create an isolated precipitate. In one embodiment, said conditionsof step e) further comprise removing said precipitated beads orsubstantially all (90% or more), so as to create a depleted supernatant.In one embodiment, the ratio of first beads to second beads in saidmixture of step a) is less than 1:10. In one embodiment, said isolatedprecipitate is contaminated with less than 5% of said first beads. Inone embodiment, said isolated precipitate is contaminated with less than2% of said first beads. In one embodiment, said isolated precipitate iscontaminated with less than 1% of said first beads. In one embodiment,said mixture of step a) further comprises a plurality of third beads. Inone embodiment, said third beads lack immobilized amplified product. Inone embodiment, said third beads comprise immobilized third amplifiedproduct, said third amplified product encoding a third nascent proteinor fragment thereof. In one embodiment, said first immobilized amplifiedproduct comprises at least a portion of a disease-related (including butnot limited to cancer-related) gene. In one embodiment, saiddisease-related gene is selected from the group consisting of the APCgene, the NF1 gene, the NF2 gene, the BRCA1 gene, the BRCA2 gene, theKras gene, and the p53 gene.

In yet another embodiment, the present invention contemplatesbead-ligand-nascent protein complexes (including but not limited tobead-ligand-nascent protein fluorescent complexes) as well as methods ofcreating and detecting a bead-ligand-nascent protein complex (includingbut not limited to a bead-ligand-nascent protein fluorescent complex).In one embodiment, the present invention contemplates a method,comprising: a) providing i) a population of template molecules, eachtemplate molecule encoding a nascent protein or protein fragment, andii) at least one surface comprising forward and reverse PCR primersattached to said surface; b) amplifying at least a portion of saidpopulation of template molecules so as to create amplified productattached to said surface; c) generating nascent protein or proteinfragment from said amplified product, said nascent protein or proteinfragment comprising an affinity tag or first epitope, and d) capturingsaid nascent protein or protein fragment on said surface via a firstligand, said first ligand attached to said bead and reactive with saidaffinity tag or first epitope. In one embodiment, said at least onesurface is on a bead. In one embodiment, the present inventioncontemplates the bead-ligand-nascent protein complex created by themethod (as a composition of matter). In one embodiment, said firstligand is attached to said bead after step b) and prior to step c). Inone embodiment, said first ligand comprises an antibody. In oneembodiment, said first ligand comprises a metal chelator. In oneembodiment, said affinity tag comprises biotin and said first ligand isselected from the group consisting of avidin and streptavidin. In oneembodiments said antibody is attached to said bead through abiotin-streptavidin linkage. In one embodiment, said amplifying of stepb) comprises i) mixing a plurality of beads in solution with saidtemplate under conditions such that at least a portion of said templatehybridizes to at least a portion of said PCR primers on at least aportion of said beads to create hybridized primers, ii) extending atleast a portion of said hybridized primers to created treated beads,iii) washing said treated beads so as to create washed beads, saidwashed beads being substantially free (e.g. 90% or more removed) oftemplate, and iv) thermally cycling said washed beads in the presence ofamplification reagents. In one embodiment, said amplification reagentscomprise a thermostable polymerase. In one embodiment, the nascentprotein or fragment thereof generated in step c) is generated in acell-free translation reaction. In one embodiment, said affinity tag isintroduced into said nascent protein during said translation reaction.In one embodiment, said antibody reacts with said first epitope on saidnascent protein. In one embodiment, the nucleic acid encoding said firstepitope is introduced during amplification in step b). In oneembodiment, said first epitope is encoded by a nucleic acid sequence ofone of said PCR primers. In one embodiment, said forward PCR primercomprises i) a sequence corresponding to a promoter, ii) a sequencecorresponding to a ribosome binding site, iii) a start codon, and iv)said sequence coding for said first epitope. In one embodiment, saidforward PCR primer further comprises v) a sequence complementary to atleast a portion of said template molecules. In one embodiment, saidtemplate sequence comprises at least a region of a gene, said geneselected from the group consisting of the APC gene, the NF1 gene, theNF2 gene, the BRCA1 gene, the BRCA2 gene, the Kras gene, the p53 gene,and the BCR-able gene. In one embodiment, said reverse PCR primercomprises i) at least one stop codon, and ii) a sequence coding for asecond epitope. In one embodiment, said first ligand is attached via aphotocleavable linker. In one embodiment, said captured nascent proteinor protein fragment of step d) is photoreleased. In one embodiment, saidcaptured nascent protein of step d) comprises a second epitope. In oneembodiment, said first epitope is an N-terminal epitope and said secondepitope is a C-terminal epitope. In one embodiment, the method furthercomprises e) reacting said captured nascent protein with a secondligand, said second ligand having affinity for said second epitope. Inone embodiment, said nascent protein or protein fragment isphotoreleased onto a non-bead surface. In one embodiment, said non-beadsurface is compatible with mass spectrometry. In one embodiment, themass of said nascent protein or protein fragment is measured by massspectrometry. In one embodiment, said bead-ligand-nascent proteincomplex is detected by flow cytometry. In one embodiment, saidbead-ligand-nascent protein complex is fluorescent. In one embodiment,said fluorescent bead-ligand-nascent protein complex is analyzed under amicroscope capable of detecting fluorescence. In one embodiment, saidfluorescent bead-ligand-nascent protein complex is analyzed by afluorescent activated cell sorter. In one embodiment, said fluorescentbead-ligand-nascent protein complex is analyzed under a microarrayreader capable of detecting fluorescence. In one embodiment, saidfluorescent bead-ligand-nascent protein complex is detected bymicrofluidics.

The present invention contemplates still other embodiments ofbead-ligand-nascent protein complexes (including but not limited tobead-ligand-nascent protein fluorescent complexes) as well as otherembodiments of methods for creating and detecting a bead-ligand-nascentprotein complex (including but not limited to a bead-ligand-nascentprotein fluorescent complex). In one embodiment, the present inventioncontemplates a method, comprising: a) providing 1) a template sequenceencoding a nascent protein or fragment thereof and 2) a surfacecomprising a PCR primer, said PCR primer comprises i) a promotersequence, ii) a ribosome binding site sequence, iii) a start codonsequence, iv) a sequence coding for a first epitope, and v) a sequencecomplementary to at least a portion of said a template sequence; b)amplifying said template so as to create amplified product immobilizedon said surface, said amplified product encoding a nascent protein orfragment thereof, and encoding said first epitope; c) attaching a firstligand capable of capturing said nascent protein or fragment thereof byreacting with said first epitope; d) generating said nascent protein orfragment thereof comprising said first epitope from said amplifiedproduct, and e) capturing said nascent protein or fragment thereof onsaid surface via said first ligand, thereby generating a surfacecomprising captured nascent protein, or fragment thereof, and amplifiedproduct coding said nascent protein or fragment thereof. In oneembodiment, said surface is a bead surface. In one embodiment, thepresent invention contemplates the bead-ligand-nascent protein complexcreated by the method (as a composition of matter). In one embodiment,said first ligand comprises an antibody. In one embodiment, said firstligand comprises a metal chelator. In one embodiment, said templatesequence comprises at least a region of a gene, said gene selected fromthe group consisting of the APC gene, the NF1 gene, the NF2 gene, theBRCA1 gene, the BRCA2 gene, the Kras gene, the p53 gene, and theBCR-able gene. In one embodiment, said first ligand is attached via aphotocleavable linker. In one embodiment, said captured nascent proteinor fragment thereof of step e) is photoreleased. In one embodiment, saidcaptured nascent protein of step e) further comprises a second epitope.In one embodiment, said first epitope is an N-terminal epitope and saidsecond epitope is a C-terminal epitope. In one embodiment, the methodfurther comprises f) reacting said captured nascent protein with asecond ligand, said second ligand having affinity for said secondepitope.

The present invention contemplates still other embodiments ofbead-ligand-nascent protein complexes (and in particular,bead-ligand-nascent protein fluorescent complexes) as well as otherembodiments of methods for creating and detecting bead-ligand-nascentprotein fluorescent complexes. In one embodiment, the present inventioncontemplates a method of creating and detecting a bead-ligand-nascentprotein fluorescent complex, comprising: a) providing 1) a templatesequence encoding a nascent protein or fragment thereof and 2) a beadcomprising first and second PCR primers, said first PCR primercomprising i) a promoter sequence, ii) a ribosome binding site sequence,iii) a start codon sequence, iv) a sequence coding for a first epitope,and v) a sequence complementary to at least a portion of said a templatesequence, and said second PCR primer comprising i) at least one stopcodon, and ii) a sequence coding for a second epitope; b) amplifyingsaid template so as to create amplified product immobilized on saidbead, said amplified product encoding a nascent protein or fragmentthereof, and encoding said first and second epitopes; c) attaching tosaid bead a first ligand capable of capturing said nascent protein orfragment thereof by reacting with said first epitope; d) generating saidnascent protein or fragment thereof comprising said first epitope fromsaid immobilized amplified product, e) capturing said nascent protein orfragment thereof on said bead via said first ligand, thereby generatinga bead-ligand-nascent protein complex; f) contacting saidbead-ligand-nascent protein complex with a second ligand capable ofbinding to said second epitope, said second ligand comprising afluorescent moiety, thereby creating a bead-ligand-nascent proteinfluorescent complex; and g) detecting said fluorescent moiety of saidbead-ligand-nascent protein fluorescent complex. The present inventioncontemplates, as a composition of matter, the bead-ligand-nascentprotein fluorescent complex made according to the above method. In oneembodiment, said first ligand comprises an antibody. In one embodiment,said bead-ligand-nascent protein complex is detected by flow cytometry.In one embodiment, said fluorescent moiety of said bead-ligand-nascentprotein fluorescent complex comprises Cy3. In one embodiment, saidfluorescent bead-ligand-nascent protein complex is detected under amicroscope capable of detecting fluorescence. In one embodiment, saidfluorescent bead-ligand-nascent protein complex is detected by afluorescent activated cell sorter. In one embodiment, said fluorescentbead-ligand-nascent protein complex is detected under a microarrayreader capable of detecting fluorescence. In one embodiment, saidfluorescent bead-ligand-nascent protein complex is detected bymicrofluidics.

DESCRIPTION OF THE FIGURES

FIG. 1A. tRNA mediated labeling, isolation by incorporated PC-biotin andphoto-release into solution of cell-free expressed proteins. Lane 1 isthe initial unbound fraction corresponding to nascent GST not bindingthe NeutrAvidin beads (wash factions were also collected and analyzedbut contained negligible quantities). Lane 2 is the negative controlelution in the absence of the proper light. Lane 3 is the photo-releasedfraction following illumination with the proper light. Lane 4 is thefraction remaining bound to the beads that was subsequently released bydenaturation of the NeutrAvidin (asterisk indicates 2× more loading togel relative to other lanes).

FIG. 1B. tRNA mediated labeling, isolation by photocleavable antibodiesand photo-release into solution of cell-free expressed proteins. Lane 1is the initial unbound fraction corresponding to nascent GST not bindingthe photocleavable antibody beads (wash factions were also collected andanalyzed but contained negligible quantities). Lane 2 is the negativecontrol elution in the absence of the proper light. Lane 3 is thephoto-released fraction following illumination with the proper light.Lane 4 is the fraction remaining bound to the beads that wassubsequently released by denaturation of the antibody (asteriskindicates 2× more loading to gel relative to other lanes).

FIG. 2A. Purity of cell-free proteins following photo-isolation byincorporated PC-biotin. Fluorescence image of electrophoretic gel(pre-staining). Lane 1 is plain SDS-PAGE gel loading buffer as anegative control. Lane 2 is the plain buffer used in the isolation as anegative control. Lane 3 is a negative control corresponding to thephoto-released fraction derived from a cell-free expression reactionwhere only the added DNA (GST gene in plasmid) was omitted. Lane 4 isthe photo-released fraction derived from a cell-free expression reactionwhere the GST DNA was included.

FIG. 2B. Purity of cell-free proteins following photo-isolation byincorporated PC-biotin. Silver stain total protein image of sameelectrophoretic gel (post-staining). Lane 1 is plain SDS-PAGE gelloading buffer as a negative control. Lane 2 is the plain buffer used inthe isolation as a negative control. Lane 3 is a negative controlcorresponding to the photo-released fraction derived from a cell-freeexpression reaction where only the added DNA (GST gene in plasmid) wasomitted. Lane 4 is the photo-released fraction derived from a cell-freeexpression reaction where the GST DNA was included. The asterisk denotesan unknown global contamination originating either in theelectrophoretic gel itself or the SDS-PAGE loading buffer but notattributable to the cell-free expressed samples or isolation process.

FIG. 3. Contact photo-transfer by incorporated PC-biotin of cell-freeexpressed, tRNA labeled and isolated proteins (antibody detection). UVlight dependence of the transfer is shown.

FIG. 4. Contact photo-transfer by incorporated PC-biotin of cell-fteeexpressed, tRNA labeled and isolated proteins (antibody detection).Comparison to separate photo-release into solution followed byapplication of the fluid elution to the activated solid microarraysurface.

FIG. 5. Contact photo-transfer by incorporated PC-biotin of cell-freeexpressed, tRNA labeled and isolated proteins. Detection via thedirectly incorporated tRNA mediated fluorescence label.

FIG. 6. Contact photo-transfer by incorporated PC-biotin of cell-freeexpressed, tRNA labeled and isolated proteins. Transfer to 3-dimensionalHydroGel matrix coated microarray substrates and detection via thedirectly incorporated tRNA mediated fluorescence label.

FIG. 7. Contact photo-transfer by incorporated PC-biotin of cell-freeexpressed, tRNA labeled and isolated proteins. Transfer from 1 micronmagnetic beads to an antibody coated surface and detection via thedirectly incorporated tRNA mediated fluorescence label.

FIG. 8. Photo-transfer by incorporated PC-biotin of cell-free expressed,tRNA labeled and isolated proteins. Transfer to the uncoated surface of96-well polystyrene microtiter plates. Detection by antibody.

FIG. 9. Photo-transfer by incorporated PC-biotin of cell-free expressed,tRNA labeled and isolated proteins. Transfer to the antibody coatedsurface of 96-well polystyrene microtiter plates. Both capture on plateand detection achieved with antibodies in a standard sandwich ELISAformat.

FIG. 10. Advanced 2 color fluorescence calcineurin-calmodulinprotein-protein interaction assays on microarrays prepared with andwithout the contact photo-transfer method.

FIG. 11A. Advanced kinase substrate profiling assays using proteins assubstrates printed to microarray surfaces by contact photo-transfer.Minus ATP negative control kinase reaction and phosphotyrosine detectionfollowed by anti-HSV total photo-transferred protein detection.

FIG. 11B. Advanced kinase substrate profiling assays using proteins assubstrates printed to microarray surfaces by contact photo-transfer.Plus ATP kinase reaction for 2 kinases followed by phosphotyrosinedetection.

FIG. 12. Contact photo-transfer from single 100 micron agarose beads byincorporated PC-biotin of cell-free expressed, tRNA labeled and isolatedproteins. Transfer to activated microarray substrates. Detection via thedirectly incorporated tRNA mediated fluorescence label and by antibody.

FIG. 13. Contact photo-transfer from single 100 micron agarose beads byincorporated PC-biotin of cell-free expressed, tRNA labeled and isolatedproteins. Transfer to activated microarray substrates. Advanced 2 colorfluorescence p53-MDM protein-protein interaction assay.

FIG. 14. Contact photo-transfer of pre-formed protein-protein complexesfrom single 100 micron agarose beads by incorporated PC-biotin. Advanced2 color fluorescence p53-MDM protein-protein interaction assay.Importantly, protein-protein complexes between MDM and p53 are formedprior to contact photo-transfer to activated microarray substrates. Boththe “bait” proteins (MDM and GST) and the p53 probe were expressed in acell-free reaction, each with appropriate tRNA mediated labels neededfor the assay.

FIG. 15A. Photo-release and contact photo-transfer of cell-freeexpressed, tRNA labeled and photocleavable antibody isolated proteins.Confirmation of successful photocleavable antibody mediated isolationand subsequent photo-release into solution.

FIG. 15B. Photo-release and contact photo-transfer of cell-freeexpressed, tRNA labeled and photocleavable antibody isolated proteins.After validation of successful photocleavable antibody mediatedisolation and the ability to photo-release into solution (see FIG. 15A),compatibility with contact photo-transfer from beads to an aldehydeactivated glass microarray substrate was also demonstrated. Detection onthe microarray substrate was via the directly incorporated tRNA mediatedfluorescence label.

FIG. 16. Photo-transfer of cell-free expressed and photocleavableantibody isolated proteins. Transfer to the nickel metal chelate coatedsurface of 96-well microtiter plates using a polyhistidine tag bindingmechanism (tag in expressed proteins). Detection of the already-boundphotocleavable antibody via a secondary antibody reporter conjugate.

FIG. 17. Contact photo-transfer of cell-free expressed, tRNA labeled andphotocleavable antibody isolated proteins. Transfer to activatedmicroarray substrates. Advanced 2 color fluorescencecalmodulin-calcineurin protein-protein interaction assay.

FIG. 18A. Preparation of photocleavable fluorescent Quantum Dotnanocrystals by conjugation to PC-biotin. Selective capture on 100micron NeutrAvidin agarose beads. Shown here is the total Quantum Dotfluorescence of the NeutrAvidin agarose bead suspension prior to washingaway any unbound Quantum Dots.

FIG. 18B. Preparation of photocleavable fluorescent Quantum Dotnanocrystals by conjugation to PC-biotin. Selective capture on 100micron NeutrAvidin agarose beads. Shown here is the Quantum Dotfluorescence bound to the NeutrAvidin agarose bead pellet afterextensive washing away of any unbound Quantum Dots and removing thefluid supernatant.

FIG. 19. Contact photo-transfer of photocleavable fluorescent QuantumDot nanocrystals from 100 micron NeutrAvidin agarose beads. Lightdependence of transfer and fluorescence emissions specificity (605 nmpeak emissions Quantum Dots).

FIG. 20. Phorbol ester (PMA) mediated protein kinase Cα (PKCα)sub-cellular translocation as measured by functional activity followingisolation with photocleavable antibodies. Cultured HeLa cells werestimulated with 200 nM PMA for 5 min and detergent fractionated into thecytosol and membrane compartments prior to isolation and purificationwith the photocleavable antibodies. The graph shows relativesub-cellular distribution of PKCα based on kinase activity of thephotocleavable antibody isolated protein.

FIG. 21. Contact photo-transfer from individually resolved beads in athin liquid film under a cover glass. A liquid suspension of 100 micronagarose beads bearing the photocleavably linked and fluorescentlylabeled protein is applied to an activated microarray substrate. Thedroplet of bead suspension is then overlaid with a circular microscopecover glass forming a thin liquid film between the cover glass and themicroarray substrate. The fluorescent protein is then contactphoto-transferred from the beads to the microarray substrate by lighttreatment through the overlaid cover glass.

FIG. 22A. Verification of binding of biotin labeled PCR amplified DNA tostreptavidin agarose beads as detected using the PicoGreen fluorescencestaining reagent selective for double stranded DNA.

FIG. 22B. Cell-free protein synthesis from expression DNA bound toagarose beads and in situ capture of the nascent proteins by PC-antibodyalso immobilized on the same agarose beads. After expression, in situprotein capture and isolation, proteins were applied to a microarraysubstrate by contact photo-transfer and the internal tRNA mediatedBODIPY-FL fluorescence labels were imaged.

FIG. 23A. Cell-free protein synthesis from expression DNA bound toagarose beads and in situ capture of the nascent proteins by PC-antibodyalso immobilized on the same agarose beads. A mixed population of beadsencoded with either GST DNA or p53 DNA were co-expressed in a singlecell-free reaction. After expression, in situ protein capture andisolation, proteins were applied to a microarray substrate by contactphoto-transfer. The microarray substrate was further probed with a Cy5labeled anti-p53 specific antibody. The internal tRNA mediated BODIPY-FLfluorescence labels as well as binding of the Cy5 labeled p53 antibodywere imaged.

FIG. 23B. Cell-free protein synthesis from expression DNA bound toagarose beads and in situ capture of the nascent proteins by PC-antibodyalso immobilized on the same agarose beads. A mixed population of beadsencoded with either GST DNA or p53 DNA were co-expressed in a singlecell-free reaction. After expression, in situ protein capture andisolation, proteins were applied to a microarray substrate by contactphoto-transfer. The microarray substrate was further probed with a Cy5labeled anti-p53 specific antibody. The internal tRNA mediated BODIPY-FLfluorescence labels as well as binding of the Cy5 labeled p53 antibodywere imaged. The images were quantified to determine the integratedfluorescence intensities for each spot for both the red (Cy5) and green(BODIPY-FL) fluorescence signals and the ratios calculated.

FIG. 24. High density arrays by contact photo-transfer from 10 micronbeads. Avidin or negative control BSA coated beads were treated withcasein that was dual labeled with PC-biotin and Cy5 (“PC-Casein”). Beadswere contact photo-transferred in a 7 mm diameter circular region. Atthis density, 4,896 spots were counted in the 7 mm circular area whichwould correspond to 242,004 spots on an entire 25×75 mm microarraysubstrate. Bead derived spots measure 13 microns diameter. +hν=Contactphoto-transfer with proper light. −hν=Negative control contact transferin the absence of proper light.

FIG. 25. Contact photo-transfer for molecular diagnostic assays.Cell-free expression from a PCR template of segment 3 of the APC geneamplified from human genomic DNA. Truncating mutations in APC are linkedto the pathogenesis of colorectal cancer. Protein based cell-freeexpression assays can be performed with N- and C-terminal tags to detectthe relative amount of truncated gene product for diagnostic purposes.Here, the tRNA mediated, BODIPY-FL internal labeling of cell-freeexpressed APC segment 3 protein is shown following contactphoto-transfer from individually resolved beads. Bead-derived microarrayfeatures of about 100 microns in diameter are created. −DNA=theprocedure performed where only the PCR derived expression DNA is omittedfrom the cell-free translation step. APC_(PCR)=the procedure performedwhere the PCR derived expression DNA for segment 3 of the human APC geneis included in the cell-free translation step.

FIG. 26A. Contact photo-transfer based microarray assays for detectionof allergen-specific IgE in human sera from allergy patients. The milkallergen casein was contact photo-transferred from beads to a microarraysubstrate and the allergen-specific IgE assay performed on themicroarray substrate. “Negative Serum” corresponds to an assay of serumfrom a non-allergic patient while “Milk Allergy Serum” corresponds to anassay of serum from a patient with a verified casein-dependant milkallergy. Both undiluted (“1×”) and 10-fold diluted (“ 1/10×”) serum wastested. The “Negative Serum” was undiluted. The “Cy5 Channel” shows thefluorescence signal from the transferred casein itself, which containeda direct Cy5 label. The “Cy3 Channel” shows detection ofallergen-specific IgE (sIgE) bound to the transferred casein frompatient sera.

FIG. 26B. Contact photo-transfer based microarray assays for detectionof allergen-specific IgE in human sera from allergy patients. The milkallergen casein was tethered to beads with a photocleavable linker andthe allergen-specific IgE assay performed directly on the beads. Thebound material on the beads was then contact photo-transferred to amicroarray substrate for signal readout. “Negative Serum” corresponds toan assay of serum from a non-allergic patient while “Milk Allergy Serum”corresponds to an assay of serum from a patient with a verifiedcasein-dependant milk allergy. The “Cy5 Channel” shows the fluorescencesignal from the transferred casein itself, which contained a direct Cy5label. The “Cy3 Channel” shows detection of allergen-specific IgE (sIgE)bound to the casein from patient sera.

FIG. 27A. Verification of binding of amine functionalized PCR primers toamine-reactive NHS ester activated agarose beads as detected using theOliGreen fluorescence staining reagent selective for single strandedDNA. The beads were prepared for the downstream purposes of solid-phasebridge PCR.

FIG. 27B. Cell-free expression of human glutathione-s-transferase A2from bead-bound DNA which was created by primer-conjugated agarose beadsused in a solid-phase bridge PCR reaction (Lane 3). Comparisons weremade to beads lacking the bound solid-phase bridge PCR primers (Lane 4)and expression reactions of human p53 and humanglutathione-s-transferase A2 using soluble plasmid DNA instead ofbead-bound DNA (Lanes 1 and 2 respectively). Arrows indicate thepositions of the fluorescently labeled expressed target protein bands onthe SDS-PAGE gel.

FIG. 28. Cell-free protein synthesis from immobilized DNA created bysolid-phase bridge PCR on agarose beads and in situ capture of thenascent proteins by PC-antibody also immobilized on the same agarosebeads. A mixed population of beads coated with primer sets to eitherγ-actin or p53 were used in a single-tube, multiplex, solid-phase bridgePCR reaction with a HeLa cell cDNA library as template. The resultantbeads, now encoded with either γ-actin DNA or p53 DNA were loaded withPC-antibody and co-expressed in a single multiplex cell-free reaction.After expression, in situ protein capture and isolation, proteins werethen applied to a microarray substrate by contact photo-transfer. Themicroarray substrate was further probed with a Cy5 labeled anti-p53specific antibody. The internal tRNA mediated BODIPY-FL fluorescencelabels (Green Fluorescence Channel) as well as binding of the Cy5labeled p53 antibody (Red Fluorescence Channel) were imaged. The figureshows the identical region of the microarray substrate in the 2different fluorescence channels. Arrows denote the p53 spots.

FIG. 29. Solid-phase bridge PCR on 7 micron diameter biotin-BSA andprimer coated beads with on-bead detection of the solid-phase bridge PCRamplicon using BODIPY-FL-dUTP labeling. Solid-phase bridge PCR reactionswere performed either minus or plus the needed DNA polymerase. The minusDNA polymerase solid-phase bridge PCR reaction provides the backgroundlevels related to bead auto-fluorescence and the BODIPY-FL-dUTP labelingreagent.

FIG. 30. 7 micron diameter plastic beads: solid-phase bridge PCR,cell-free protein expression, in situ protein capture, antibody probingand isolation of the antibody targeted bead sub-population. Solid-phasebridge PCR beads carrying amplified and expressible DNA for the p53 andGST genes were separately cell-free expressed, with in situ proteincapture onto the same beads, using a bead-bound rabbit anti-HSV antibodyagainst a common epitope tag in both proteins. Beads were then mixed at1% p53 beads and 99% GST beads followed by probing the mixed beads witha mouse monoclonal anti-p53 antibody. The p53 bead sub-population,targeted by the anti-p53 antibody, was then purified using 1 micronmagnetic particles which were coated with an anti-[mouse IgG]species-specific secondary antibody (magnetic particles not visible infigure). The un-separated and purified beads were embedded in apolyacrylamide film on a microscope slide and the fluorescence beadlabels then imaged. The same regions of the microscope slide were imagedin both the green fluorescence channel (GST beads) and red fluorescencechannel (p53 beads).

FIG. 31A. Contact photo-transfer of cell-free expressed peptides ontoglass microarray slides followed by mass spectrometry analysis(MALDI-TOF). FLAG epitope tagged CT61 and CT64 test peptides werecell-free expressed and isolated on beads that carry a photocleavablylinked and fluorescently labeled anti-FLAG antibody. The beads were thenused to contact photo-transfer the peptide-antibody complexes to anepoxy activated-microarray slide. The microarray slide was first imagedfluorescently. The minus DNA (−DNA) negative control corresponds to aparallel sample differing only by omission of the expression DNA fromthe cell-free reaction.

FIG. 31B. Contact photo-transfer of cell-free expressed peptides ontoglass microarray slides followed by mass spectrometry analysis(MALDI-TOF). FLAG epitope tagged CT61 and CT64 test peptides werecell-free expressed and isolated on beads that carry a photocleavablylinked and fluorescently labeled anti-FLAG antibody. The beads were thenused to contact photo-transfer the peptide-antibody complexes to anepoxy activated microarray slide. After fluorescence imaging (see FIG.31A), the microarray slide was then subjected to MALDI-TOF massspectrometric analyses. The minus DNA (−DNA) negative controlcorresponds to a parallel sample differing only by omission of theexpression DNA from the cell-free reaction. The black spectrum is CT61,the dark gray spectrum CT64 and the light gray is the minus DNA (−DNA).

FIG. 32A. Contact photo-transfer of DNA from beads to an activatedmicroarray slide. DNA was either labeled with PC-biotin (+PCB) or leftunlabeled (—PCB). NeutrAvidin agarose beads were then used to capturethe DNA. The beads were subsequently used for contact photo-transfer(see FIG. 32B). Prior to contact photo-transfer, DNA binding to thebeads was first verified using either the ssDNA fluorescence stainOliGreen (upper panels) or a Cy5 fluorescence labeled complementaryoligonucleotide probe (lower panels). The bead pellets (Beads) wereimaged directly in 0.5 mL micro-centrifuge tubes.

FIG. 32B. Contact photo-transfer of DNA from beads to an activatedmicroarray slide. DNA was labeled with PC-biotin and NeutrAvidin agarosebeads were then used to capture the DNA. The beads were subsequentlyused for contact photo-transfer. Beads loaded with the PC-biotin labeledDNA, but not previously stained with OliGreen or a complementaryoligonucleotide probe (as done in FIG. 32A), were used for contactphoto-transfer. Contact photo-transfer was performed with (+Light) orwithout (−Light) the proper light illumination. After contactphoto-transfer onto epoxy activated microarray slides, the slides wereprobed with either a biotin labeled complementary oligonucleotidefollowed by a NeutrAvidin-Cy5 conjugate (upper panels) or a directly Cy5labeled complementary oligonucleotide (lower panels).

FIG. 33A. Effective single template molecule solid-phase bridge PCR:Amplicon detection through fluorescence dUTP labeling during the PCRreaction. Conditions were targeted to achieve solid-phase bridge PCRamplification of one or a few template DNA molecules per bead, onagarose beads that were covalently conjugated to both the forward andreverse PCR primers. The added template solution was a mixture of 50%human GST A2 (gene fragment) and 50% human p53 (gene fragment), withboth gene fragments flanked by universal sequences to which thesolid-phase primers were directed. Following template capture(annealing) and extending the primers only once in the presence of DNApolymerase, any free or hybridized template DNA was washed from thebeads in 0.1 N NaOH, leaving only covalently attached unused andextended primers. The beads were then subjected to full PCRthermocycling in a high-fidelity PCR reaction mixture, to facilitatesolid-phase bridge PCR amplification. Labeling of the PCR amplicon(product) on the beads was achieved by using BODIPY-FL conjugatedfluorescent dUTP (green) in the PCR reaction mix. Following solid-phasebridge PCR, the beads were washed and then probed with a NeutrAvidin-Cy5fluorescent conjugate (red), which binds to biotin labels which wereuniformly covalently attached directly to the agarose bead surfaceduring the earlier primer attachment procedure; thus detecting all beadsregardless of the presence of PCR amplicon (red). The beads were washedagain then embedded in a polyacrylamide film on a standard microscopeslide for imaging in a fluorescence microarray reader. The imagescorrespond to 2-color fluorescence image overlays (same image contrastsettings) of the minus template (−Template) and plus template (180 and1,800 attomoles of template per μL of agarose bead volume) solid-phasebridge PCR reaction samples. In the 2-color fluorescence image overlay,a yellow-orange color indicates the presence of both the green and redsignals, however, at higher amplicon levels, the green signal masks thered in the overlaid images.

FIG. 33B. Effective single template molecule solid-phase bridge PCR:Amplicon detection through fluorescence dUTP labeling during the PCRreaction. Conditions were targeted to achieve solid-phase bridge PCRamplification of one or a few template DNA molecules per bead, onagarose beads that were covalently conjugated to both the forward andreverse PCR primers. The added template solution was a mixture of 50%human GST A2 (gene fragment) and 50% human p53 (gene fragment), withboth gene fragments flanked by universal sequences to which thesolid-phase primers were directed. Following template capture(annealing) and extending the primers only once in the presence of DNApolymerase, any free or hybridized template DNA was washed from thebeads in 0.1 N NaOH, leaving only covalently attached unused andextended primers. The beads were then subjected to full PCRthermocycling in a high-fidelity PCR reaction mixture, to facilitatesolid-phase bridge PCR amplification. Labeling of the PCR amplicon(product) on the beads was achieved by using BODIPY-FL conjugatedfluorescent dUTP (green) in the PCR reaction mix. Following solid-phasebridge PCR, the beads were washed and then probed with a NeutrAvidin-Cy5fluorescent conjugate (red), which binds to biotin labels which wereuniformly covalently attached directly to the agarose bead surfaceduring the earlier primer attachment procedure; thus detecting all beadsregardless of the presence of PCR amplicon (red). The beads were washedagain then embedded in a polyacrylamide film on a standard microscopeslide for imaging in a fluorescence microarray reader. More than 350beads per each sample permutation were quantified by computer-assistedimage analysis and the green:red signal ratios were calculated andplotted. Permutations were the minus template (−Template) and plustemplate (180 and 1,800 attomoles of template per μL of agarose beadvolume) solid-phase bridge PCR reaction samples. The Y-axis is thegreen:red signal ratio and the X-axis is the bead number. The red lineindicates the cut-off, at or above which the beads are scored as “strongpositives”.

FIG. 34A. Effective single template molecule solid-phase bridge PCR:Amplicon detection through fluorescence dUTP labeling during the PCRreaction. Conditions were targeted to achieve solid-phase bridge PCRamplification of one or a few template DNA molecules per bead, onagarose beads that were covalently conjugated to both the forward andreverse PCR primers. The added template solution was a mixture of 75%human GST A2 (gene fragment) and 25% human p53 (gene fragment), withboth gene fragments flanked by universal sequences to which thesolid-phase primers were directed. Following template capture(annealing) and extending the primers only once in the presence of DNApolymerase, any free or hybridized template DNA was washed from thebeads in 0.1 N NaOH, leaving only covalently attached unused andextended primers. The beads were then subjected to full PCRthermocycling in a high-fidelity PCR reaction mixture, to facilitatesolid-phase bridge PCR amplification. Labeling of the PCR amplicon(product) on the beads was achieved by using BODIPY-FL conjugatedfluorescent dUTP (green) in the PCR reaction mix. Following solid-phasebridge PCR, the beads were washed and then probed with a NeutrAvidin-Cy5fluorescent conjugate (red), which binds to biotin labels which wereuniformly covalently attached directly to the agarose bead surfaceduring the earlier primer attachment procedure; thus detecting all beadsregardless of the presence of PCR amplicon (red). The beads were washedagain and embedded in a polyacrylamide film on a standard microscopeslide for imaging in a fluorescence microarray reader. The imagescorrespond to 2-color fluorescence image overlays (same image contrastsettings) of the minus template (−Template) and plus template (18 and180 attomoles of template per μL of agarose bead volume) solid-phasebridge PCR reaction samples. In the 2-color fluorescence image overlay,a yellow-orange color indicates the presence of both the green and redsignals, however, at higher amplicon levels, the green signal masks thered in the overlaid images.

FIG. 34B. Effective single template molecule solid-phase bridge PCR:Amplicon detection through fluorescence dUTP labeling during the PCRreaction. Conditions were targeted to achieve solid-phase bridge PCRamplification of one or a few template DNA molecules per bead, onagarose beads that were covalently conjugated to both the forward andreverse PCR primers. The added template solution was a mixture of 75%human GST A2 (gene fragment) and 25% human p53 (gene fragment), withboth gene fragments flanked by universal sequences to which thesolid-phase primers were directed. Following template capture(annealing) and extending the primers only once in the presence of DNApolymerase, any free or hybridized template DNA was washed from thebeads in 0.1 N NaOH, leaving only covalently attached unused andextended primers. The beads were then subjected to full PCRthermocycling in a high-fidelity PCR reaction mixture, to facilitatesolid-phase bridge PCR amplification. Labeling of the PCR amplicon(product) on the beads was achieved by using BODIPY-FL conjugatedfluorescent dUTP (green) in the PCR reaction mix. Following solid-phasebridge PCR, the beads were washed and then probed with a NeutrAvidin-Cy5fluorescent conjugate (red), which binds to biotin labels which wereuniformly covalently attached directly to the agarose bead surfaceduring the earlier primer attachment procedure; thus detecting all beadsregardless of the presence of PCR amplicon (red). The beads were washedagain and embedded in a polyacrylamide film on a standard microscopeslide for imaging in a fluorescence microarray reader. More than 350beads per each sample permutation were quantified by computer-assistedimage analysis and the green:red signal ratios were calculated andplotted. Permutations were the minus template (−Template) and plustemplate (18 and 180 attomoles of template per μL of agarose beadvolume) samples. The Y-axis is the green:red signal ratio and the X-axisis the bead number. The red line indicates the cut-off, at or abovewhich the beads are scored as “strong positives”.

FIG. 35. Effective single template molecule solid-phase bridge PCR:Amplicon detection by dual oligonucleotide hybridization probing.Conditions were targeted to achieve solid-phase bridge PCR amplificationof one or a few template DNA molecules per bead, on agarose beads thatwere covalently conjugated to both the forward and reverse PCR primers.The added template solution was a mixture of 75% human GST A2 (genefragment) and 25% human p53 (gene fragment), with both gene fragmentsflanked by universal sequences to which the solid-phase primers weredirected. Template was added at 180 attomoles per μL of agarose beadvolume. Following template capture (annealing) and extending the primersonly once in the presence of DNA polymerase, any free or hybridizedtemplate DNA was washed from the beads in 0.1 N NaOH, leaving onlycovalently attached unused and extended primers. The beads were thensubjected to full PCR thermocycling in a high-fidelity PCR reactionmixture, to facilitate solid-phase bridge PCR amplification. Followingsolid-phase bridge PCR, the beads were simultaneouslyhybridization-probed with gene-specific complementary oligonucleotidesthat were fluorescently labeled. The beads were embedded in apolyacrylamide film on a standard microscope slide for imaging in afluorescence microarray reader. The main left and right image panelscorrespond to 2-color fluorescence image overlays (same image contrastsettings) of the minus template (−Template) and plus template(+Template) solid-phase bridge PCR reaction samples respectively,following hybridization-probing of the beads. Human GST A2 PCR productwas detected on the beads via the Cy3 fluorophore (green) attached tothe gene-specific hybridization probe and the human p53 PCR product viathe Cy5 fluorophore (red) attached to the gene-specific hybridizationprobe. In the 2-color fluorescence image overlay, a yellow-orange colorindicates the presence of both the green and red signals. The inset boxin the main left panel (−Template) shows the presence of beads (presentthroughout entire main left panel), visible in this selected region onlyby their weak auto-fluorescence at extremely high image contrastsettings. The inset box in the main right panel (+Template) shows thenon-overlaid green and red fluorescence images of the selected boxedregion (circular outlines denote the position of beads both visible andnot visible in that particular fluorescence channel).

FIG. 36A. Effective single template molecule solid-phase bridge PCR:Titration of template ratios and amplicon detection by dualoligonucleotide hybridization probing. Conditions were targeted toachieve solid-phase bridge PCR amplification of only one or a fewtemplate DNA molecules per bead, on agarose beads that were covalentlyconjugated to both the forward and reverse PCR primers. The addedtemplate solution was a mixture of human p53 (gene fragment) and humanGST A2 (gene fragment), with both gene fragments flanked by universalsequences to which the solid-phase primers were directed. Ratios ofhuman p53 to human GST A2 within the added template solution were 50:50,75:25 and 95:5. Template was added at 180 attomoles per μL of agarosebead volume or template DNA was omitted from the solid-phase bridge PCRreaction as a negative control (−Template). Following template capture(annealing) and extending the primers only once in the presence of DNApolymerase, any free or hybridized template DNA was washed from thebeads in 0.1 N NaOH, leaving only covalently attached unused andextended primers. The beads were then subjected to full PCRthermocycling in a high-fidelity PCR reaction mixture, to facilitatesolid-phase bridge PCR amplification. Following solid-phase bridge PCR,the beads were simultaneously hybridization-probed with gene-specificcomplementary oligonucleotides that were fluorescently labeled. Thebeads were embedded in a polyacrylamide film on a standard microscopeslide for imaging in a fluorescence microarray reader. The upper row ofimage panels correspond to 3-color fluorescence image overlays. Humanp53 PCR product was detected on the beads via the Cy5 fluorophore (red)attached to the gene-specific hybridization probe and the human GST A2PCR product via the Cy3 fluorophore (green) attached to thegene-specific hybridization probe. The blue signal is a total beadfluorescence stain that is independent of the presence or absence of PCRproduct, thereby allowing detection of all beads. In the 3-colorfluorescence image overlay, a yellow-orange color indicates the presenceof both the green and red signals. The lower 2 rows of image panels are2-color fluorescence image overlays, with either the red (human p53) orgreen (human GST A2) fluorescence images turned off (i.e. omitted fromimage overlay).

FIG. 36B. Effective single template molecule solid-phase bridge PCR:Titration of template ratios and amplicon detection by dualoligonucleotide hybridization probing. Conditions were targeted toachieve solid-phase bridge PCR amplification of only one or a fewtemplate DNA molecules per bead, on agarose beads that were covalentlyconjugated to both the forward and reverse PCR primers. The addedtemplate solution was a mixture of human p53 (gene fragment) and humanGST A2 (gene fragment), with both gene fragments flanked by universalsequences to which the solid-phase primers were directed. Ratios ofhuman p53 to human GST A2 within the added template solution were 50:50,75:25 and 95:5. Template was added at 180 attomoles per μL of agarosebead volume or template DNA was omitted from the solid-phase bridge PCRreaction as a negative control (−Template). Following template capture(annealing) and extending the primers only once in the presence of DNApolymerase, any free or hybridized template DNA was washed from thebeads in 0.1 N NaOH, leaving only covalently attached unused andextended primers. The beads were then subjected to full PCRthermocycling in a high-fidelity PCR reaction mixture, to facilitatesolid-phase bridge PCR amplification. Following solid-phase bridge PCR,the beads were simultaneously hybridization-probed with gene-specificcomplementary oligonucleotides that were fluorescently labeled. Thebeads were embedded in a polyacrylamide film on a standard microscopeslide for imaging in a fluorescence microarray reader. Beads werequantified and scored. Beads were scored positive if the signal to noiseratio was 0:1. For each sample permutation, the p53 and GST A2 positivescores are plotted as a percentage of the total positive scores.

FIG. 37. Effective single template molecule solid-phase bridge PCR:Multiplexed cell-free expression with in situ protein capture, contactphoto-transfer of the expressed protein and antibody detection.Conditions were targeted to achieve solid-phase bridge PCR amplificationof one or a few template DNA molecules per bead, on agarose beads thatwere covalently conjugated to both the forward and reverse PCR primers.The added template solution was a mixture of 75% human GST A2 (genefragment) and 25% human p53 (gene fragment), with both gene fragmentsflanked by universal sequences to which the solid-phase primers weredirected. Template was added at 180 attomoles per μL of agarose beadvolume. Template DNAs also contained sequences necessary to supportcell-free protein expression of the gene fragments in addition to commonN- and C-terminal antibody epitope tags. Following template capture(annealing) and extending the primers only once in the presence of DNApolymerase, any free or hybridized template DNA was washed from thebeads in 0.1 N NaOH, leaving only covalently attached unused andextended primers. The beads were then subjected to full PCRthermocycling in a high-fidelity PCR reaction mixture, to facilitatesolid-phase bridge PCR amplification. Following solid-phase bridge PCR,the beads were uniformly coated with a photocleavable antibody againstthe common N-terminal FLAG epitope tag. The beads were then used in amultiplexed cell-free expression reaction with in situ protein capture,whereby expressed proteins are captured simultaneously onto their parentbeads, as they are produced from the bead-bound solid-phase bridge PCRproduct. Contact photo-transfer was then performed from the beads andthe resultant random microarray probed with fluorescent antibodies. Theleft and right image panels correspond to 2-color fluorescence imageoverlays (same image contrast settings) of the minus template(−Template) and plus template (+Template) solid-phase bridge PCRreaction samples, respectively, following expression, contactphoto-transfer and fluorescence antibody probing. Although arepresentative region is shown, approximately 60-100 spots were analyzedin each of the 2 samples. The green signal is the detection of thephoto-transferred FLAG antibody, and shows all spots, regardless of thepresence of detectible expressed protein. The red signal shows detectionof the common VSV epitope tag present at the C-terminal of both thehuman GST A2 and human p53 gene fragments. In the 2-color fluorescenceimage overlay, a yellow-orange color indicates the presence of both thegreen and red signals.

FIG. 38. Effective single template molecule solid-phase bridge PCR:Multiplexed cell-free expression with in situ protein capture, on-beadantibody detection and flow cytometry. Conditions were targeted toachieve solid-phase bridge PCR amplification of one or a few templateDNA molecules per bead, on agarose beads that were covalently conjugatedto both the forward and reverse PCR primers. The added template solutionwas a mixture of 75% human GST A2 (gene fragment) and 25% human p53(gene fragment), with both gene fragments flanked by universal sequencesto which the solid-phase primers were directed. Template was added at180 attomoles per μL of agarose bead volume. Template DNAs alsocontained sequences necessary to support cell-free protein expression ofthe gene fragments in addition to common N- and C-terminal antibodyepitope tags. Following template capture (annealing) and extending theprimers only once in the presence of DNA polymerase, any free orhybridized template DNA was washed from the beads in 0.1 N NaOH, leavingonly covalently attached unused and extended primers. The beads werethen subjected to full PCR thermocycling in a high-fidelity PCR reactionmixture, to facilitate solid-phase bridge PCR amplification. Followingsolid-phase bridge PCR, the beads were uniformly coated with aphotocleavable antibody against the common N-terminal FLAG epitope tag.The beads were then used in a multiplexed cell-free expression reactionwith in situ protein capture, whereby expressed proteins are capturedsimultaneously onto their parent beads, as they are produced from thebead-bound solid-phase bridge PCR product. The beads were then probedwith a Cy3 labeled antibody against the common VSV epitope tag presentat the C-terminal of both the human GST A2 and human p53 gene fragments.The beads were then analyzed by flow cytometry. The upper left and upperright image panels correspond to the minus template (−Template) and plustemplate (+Template) solid-phase bridge PCR reaction samplesrespectively, following expression, fluorescence antibody probing andflow cytometry analysis. “Positive Control” refers to a sample which didnot involve solid-phase bridge PCR, but instead expression was fromsoluble PCR derived DNA with protein capture onto FLAG antibody coatedagarose beads as with the other samples. The Y-axis corresponds to theside-scatter (bead detection regardless of fluorescence signal) and theX-axis the fluorescence signal intensity from the Cy3 labeled VSVantibody probe. The values denoted in the lower corners of each quadrantindicate the percent of beads falling within that quadrant.

FIG. 39A. Microarray protein truncation test on the APC gene associatedwith colorectal cancer: Contact photo-transfer and fluorescence antibodydetection. A segment of the human APC gene was amplified by standardsolution-phase PCR on cell-line genomic DNA, using gene-specific PCRprimers. Non-native DNA sequences necessary for cell-free proteinexpression and epitope tag detection were also incorporated (added) viathe PCR primers, by way of the non-hybridizing portions of the primers.The DNA was then cell-free expressed in a coupledtranscription/translation rabbit reticulocyte system. Followingexpression, proteins were captured on agarose beads coated with aphotocleavable antibody against the common N-terminal HSV bindingepitope tag. Beads were then used for contact photo-transfer and theresultant random microarray probed simultaneously with fluorescentlylabeled antibodies against the N- and C-terminal detection epitope tags.The images above are 2-color fluorescence overlays, whereby the greencorresponds to the N-terminal detection epitope tag probed with ananti-VSV antibody labeled with the Cy3 fluorophore and the redcorresponds to the C-terminal detection epitope tag probed with ananti-p53 antibody labeled with the Cy5 fluorophore. In the 2-colorfluorescence image overlay, a yellow-orange color indicates the presenceof both the green and red signals. “APC WT” refers to a 100% wild-typesample derived from cell-line DNA lacking any mutations in the APC genesegment. “APC Mutant” refers to a 100% mutant sample derived fromcell-line DNA containing a truncation mutation within the APC genesegment tested (i.e. nonsense mutation to stop codon). “−DNA” refers toa negative control, identical to the other samples except that only theDNA was omitted from the cell-free expression reaction.

FIG. 39B. Microarray protein truncation test on the APC gene associatedwith colorectal cancer: Contact photo-transfer and fluorescence antibodydetection. A segment of the human APC gene was amplified by standardsolution-phase PCR on cell-line genomic DNA, using gene-specific PCRprimers. Non-native DNA sequences necessary for cell-free proteinexpression and epitope tag detection were also incorporated (added) viathe PCR primers, by way of the non-hybridizing portions of the primers.The DNA was then cell-free expressed in a coupledtranscription/translation rabbit reticulocyte system. Followingexpression, proteins were captured on agarose beads coated with aphotocleavable antibody against the common N-terminal HSV bindingepitope tag. Beads were then used for contact photo-transfer and theresultant random microarray probed simultaneously with fluorescentlylabeled antibodies against the N- and C-terminal detection epitope tags.Each spot was quantified and the C-terminal to N-terminal ratio (C:NRatio) calculated. “APC WT” refers to a 100% wild-type sample derivedfrom cell-line DNA lacking any mutations in the APC gene segment. “APCMutant” refers to a 100% mutant sample derived from cell-line DNAcontaining a truncation mutation within the APC gene segment tested(i.e. nonsense mutation to stop codon). “−DNA” refers to a negativecontrol, identical to the other samples except that only the DNA wasomitted from the cell-free expression reaction. All spots were averagedfor each sample permutation (n>300), the data were normalized to set theC:N ratio of the APC WT to 100% and the data then plotted. In the bargraph, the error bars represent the standard deviation.

FIG. 40. Solid-phase bridge PCR on the APC gene associated withcolorectal cancer: Cell-free protein expression, contact photo-transferand fluorescence antibody detection. The solid-phase bridge PCR templateDNA was first prepared by amplifying a segment of the human APC geneusing standard solution-phase PCR on cell-line genomic DNA, withgene-specific PCR primers. These PCR primers also serve to introduce(add) a portion of the non-native DNA sequences needed for cell-freeprotein expression and epitope tag detection, via the non-hybridizingportion of the primers. Next, a universal forward and reverse PCR primerset, directed against these added non-native sequences, was covalentlyconjugated to agarose beads and used for solid-phase bridge PCRamplification of the aforementioned template DNA. The solid-phaseuniversal primers also serve to introduce (add) the remaining portion ofnon-native DNA sequences necessary for cell-free expression and epitopetag detection. For the solid-phase bridge PCR, the template DNA wascaptured (annealed) onto the beads in non-limiting amounts, and was amixture of 75% wild-type APC and 25% mutant APC, containing a truncationmutation within the APC gene segment tested (i.e. nonsense mutation tostop codon). Following solid-phase bridge PCR, the beads were uniformlycoated with a photocleavable antibody against the common N-terminal HSVbinding epitope tag. The beads were then used in a cell-free proteinexpression reaction, whereby proteins expressed from the bead-boundsolid-phase bridge PCR product are then captured onto the beads via thephotocleavable HSV antibody. Beads were then used for contactphoto-transfer and the resultant random microarray probed simultaneouslywith fluorescently labeled antibodies against the N- and C-terminaldetection epitope tags. “Anti-p53-Cy5” denotes results from theC-terminal detection epitope tag (p53) probed using a p53 antibodylabeled with the Cy5 fluorophore. “Anti-VSV-Cy3” denotes results fromthe N-terminal detection epitope tag (VSV) probed using a VSV antibodylabeled with the Cy3 fluorophore (same region of microarray).“+Template” refers to the test sample, where the appropriate templateDNA was indeed added to the solid-phase bridge PCR reaction. “−Template”refers to a negative control, identical to the test sample except thatonly the template DNA was omitted from the solid-phase bridge PCRreaction.

FIG. 41. Effective single template molecule solid-phase bridge PCR onthe APC gene associated with colorectal cancer: Validation of effectiveamplification of single template molecules per bead using 2 templatespecies and a single-base extension reaction as the ultimate assay. Thesolid-phase bridge PCR template DNA was first prepared by amplifying asegment of the human APC gene using standard solution-phase PCR oncell-line genomic DNA, with gene-specific PCR primers. These PCR primersalso serve to introduce (add) a portion of the non-native DNA sequencesneeded for cell-free protein expression and epitope tag detection, viathe non-hybridizing portion of the primers. Next, a universal forwardand reverse PCR primer set, directed against these added non-nativesequences, was covalently conjugated to agarose beads and used forsolid-phase bridge PCR amplification of the aforementioned template DNA.The solid-phase universal primers also serve to introduce (add) theremaining portion of non-native DNA sequences necessary for cell-freeexpression and epitope tag detection. For the solid-phase bridge PCR,the template DNA was initially added at a ratio of roughly 1 moleculeper bead. The initially added template was a mixture of 50% wild-typeAPC and 50% mutant APC, containing a truncation mutation within the APCgene segment tested (i.e. nonsense mutation to stop codon). Followingsolid-phase bridge PCR, the beads were subjected to a fluorescence basedsingle-base extension (SBE) reaction to distinguish the single-basechange between wild-type and mutant APC amplicons on the beads. The toppair of image panels indicates the minus template sample permutationwhile the bottom pair of image panels the plus template sample. The topimage in each pair corresponds to the fluorescein fluorescence channeland hence the binding of the fluorescein labeled SBE probe. The insetbox shows the presence of beads in the minus template sample, visibleonly at extremely high image intensity settings via their extremely weakauto-fluorescence. The bottom image in each pair corresponds to a2-color fluorescence image overlay of the Cy3 (wild-type extensionproduct; green) and the Cy5 (mutant extension product; red) fluorescencechannels. Arrows indicate selected beads for which the green:red signalratios are shown.

FIG. 42. Multiplexing methylation specific PCR (MSP) using solid-phasebridge PCR on beads: Application to the colorectal cancer associatedmarkers vimentin and RASSF2A. As a model system, the solid-phase bridgePCR template DNA was first prepared by solution-phase MSP amplificationof regions of bisulfite treated wild-type human genomic DNAcorresponding to the vimentin and RASSF2A markers associated withcolorectal cancer. Next, primers directed at the key bisulfite convertedsequences (regions that are unmethylated at CpG islands in the wild-typeand methylated in the disease state) were covalently conjugated toagarose beads and used for solid-phase bridge PCR amplification of theaforementioned template DNA. Two primer bead species, for vimentin andRASSF2A, were prepared separately. Following solid-phase bridge PCRamplification on the beads, the amplicon was detected on the beads bydual probing with fluorescently labeled complementary oligonucleotides.The vimentin probe was labeled with Cy3 (green) and the RASSF2A with Cy5(red). Image panels marked “Multiplex” correspond to samples where bothprimer bead species were used in the solid-phase bridge PCR reaction ata 50:50 ratio. Image panels marked “Single-Plex” correspond to sampleswere only one bead species was used. “−Template” indicates that only thetemplate DNA was omitted from the solid-phase bridge PCR reaction(negative control). When template was included, “Multiplex” samplesreceived both templates while “Single-Plex” samples received only thecorresponding template.

FIG. 43. Solid-Phase bridge PCR on the APC gene associated withcolorectal cancer: Direct use of genomic DNA templates in thesolid-phase bridge PCR reaction. Solid-Phase bridge PCR was performed onagarose beads using fragmented genomic DNA as a template. To do so,agarose beads covalently conjugated to an APC gene-specific primer setwere prepared. Non-hybridizing regions of the primers also incorporateall necessary untranslated regions for downstream cell-free proteinexpression and epitope tag detection of the N- and C-terminals of theexpressed protein. Supplementation with magnesium and DNA polymerase inthe solid-phase bridge PCR reaction was tested. Following completion ofthe solid-phase bridge PCR reaction, all beads, which also containconjugated biotin moieties, were stained with streptavidin Alexa Fluor488 (green). Beads were then probed (hybridized) with a complementaryCy5 labeled oligonucleotide direct against internal sequences of the APCsolid-phase bridge PCR amplicon (red). 2-color overlays of the green andred fluorescence images are presented.

FIG. 44A. Solid-Phase bridge PCR on 6 micron diameter, non-porous,fluorescently bar-coded plastic beads from Luminex Corporation:Verification of primer attachment to the beads prior to solid-phasebridge PCR. Both forward and reverse primers directed against a templatederived from the human APC gene were covalently attached to the beads bytheir 5′ ends for later use of the beads in solid-phase bridge PCRreactions. Two types of fluorescently bar-coded beads from LuminexCorporation (Austin, Tex.) were tested, xMAP and SeroMAP, which weredesigned for multiplexed assays (e.g. multiplexed SNP or immunoassays).To verify successful primer attachment to the beads, the beads werestained with the fluorescent single-stranded DNA detection agentOliGreen (Invitrogen Corporation, Carlsbad, Calif.). Stained beadpellets (˜0.125 μL actual bead pellet volume) were fluorescently imageddirectly in 0.5 mL thin-wall polypropylene PCR tubes. “+Primer”indicates beads that were chemically conjugated to the primers, while“−Primer” indicates beads that were subjected to the chemicalconjugation procedure, but omitting only the primers from the reaction.The image was artificially colorized in Pseudocolor using ImageQuantquantitative image analysis software (Molecular Dynamics; AmershamBiosciences Corp., Piscataway, N.J.) to better show differences influorescence intensity and the corresponding scale is shown.

FIG. 44B. Solid-Phase bridge PCR on 6 micron diameter, non-porous,fluorescently bar-coded plastic beads from Luminex Corporation:Detection of the solid-phase bridge PCR amplicon on the beads bybiotin-dUTP labeling. Both forward and reverse primers directed againsta template derived from the human APC gene were covalently attached tothe beads by their 5′ ends. Solid-phase bridge PCR was performed in thepresence of biotin-16-dUTP for labeling of the amplicon. Followingsolid-phase bridge PCR, amplicon was detected on the beads viachemiluminescence using a NeutrAvidin-HRP conjugate. The data wasplotted in bar chart form and RLU represents the Relative LuminescenceUnits (arbitrary units). “Plus Template” refers to samples wheretemplate DNA was included in the solid-phase bridge PCR reaction. “MinusTemplate” refers to parallel samples whereby only the template DNA wasomitted from the solid-phase bridge PCR reaction, but were otherwiseidentical to the “Plus Template” samples. Two types of fluorescentlybar-coded beads from Luminex Corporation (Austin, Tex.) were tested,xMAP and SeroMAP, which were designed for multiplexed assays (e.g.multiplexed SNP or immunoassays).

FIG. 45. Solid-Phase bridge PCR for detection of the bisulfite convertedwild-type vimentin DNA marker directly from genomic DNA: Applications incolorectal cancer diagnosis. Template for the solid-phase bridge PCRreaction was wild-type human genomic DNA that was fragmented andbisulfite converted to distinguish between methylated and unmethylatedsequences. Next, primers directed at the key bisulfite convertedsequences in the vimentin marker (regions that are unmethylated at CpGislands in the wild-type and methylated in the disease state) werecovalently conjugated to agarose beads and used for solid-phase bridgePCR amplification of the aforementioned template DNA. Followingsolid-phase bridge PCR amplification on the beads, the amplicon wasdetected on the beads by probing with a fluorescently labeledcomplementary oligonucleotide. This vimentin probe was labeled with Cy3fluorescence. “+gDNA Template” indicates when the fragmented andbisulfite converted genomic DNA template was added to the solid-phasebridge PCR reaction. “−Template” indicates that only the template DNAwas omitted from the solid-phase bridge PCR reaction (negative control).

FIG. 46. Solid-Phase bridge PCR followed by cell-free expression with insitu protein capture on PC-antibodies: Background reduction in massspectrometry analysis by subsequent photo-release. Solid-phase bridgePCR was performed on beads to amplify a segment of the BCR-ABL tyrosinekinase domain (designated Segment 1 in this Example). The solid-phasebridge PCR primers additionally incorporated sequences necessary forcell-free protein expression as well as an N-terminal FLAG epitope tag.Following solid-phase bridge PCR, a photocleavable antibody(PC-antibody), directed against the FLAG epitope tag, was bound to thebeads and the beads used to mediate cell-free protein expression. Theexpressed peptide was in situ captured onto the same beads, during theexpression reaction. Following extensive washing, the captured peptidewas eluted from the beads either by denaturation of the PC-antibody orby photo-release of the PC-antibody. The eluted peptide was thenanalyzed by MALDI-TOF mass spectrometry. The asterisk and “2×” in thefigure denote the plus matrix adduct and doubly charged versions of theSegment 1 peptide, respectively, and hence are not contaminants.

FIG. 47. Solid-phase bridge PCR followed by cell-free expression andmass spectrometry analysis: Multiplex cell-free expression. A singlemultiplexed solid-phase bridge PCR reaction was performed on 6 differentsegments of the BCR-ABL transcript involved in Chronic Myeloid Leukemia(CML). The solid-phase bridge PCR primers additionally incorporatedsequences needed for efficient cell-free protein expression and epitopetagging. A single multiplexed cell-free protein expression reaction wasthen performed using the post solid-phase bridge PCR beads as thetemplate DNA source (bead mixture of all 6 segments). Followingexpression, the crude peptides were affinity co-purified via theircommon N-terminal FLAG epitope tag and analyzed by MALDI-TOF massspectrometry. Peptide peaks in the mass spectrum are labeled with theircorresponding segment number. All segments were clearly identified witha 1 Dalton mass accuracy. The +75 peak was putatively identified as aSNP of Segment 7.

FIG. 48. Affinity purification of cell-free expressed peptides onto anagarose bead affinity resin followed by mass spectrometry detection fromsingle beads. A conventional solution-phase PCR reaction was performedon a segment of the APC gene involved in colorectal cancer. The PCRprimers additionally incorporated sequences needed for efficientcell-free protein expression and epitope tagging. The PCR product DNAwas then used to mediate cell-free protein expression. Followingexpression, the crude peptide was affinity purified via its N-terminalFLAG epitope tag and analyzed by MALDI-TOF mass spectrometry. The labelsin parenthesis correspond to the signal intensity of the expected targetpeak (arbitrary units). The asterisks indicate the minor plus matrixadduct of the target peak. Spectra from 3 different individual 100micron diameter agarose beads are shown.

DETAILED DESCRIPTION OF THE INVENTION

The present invention features methods and compositions for theproduction of biomolecules on beads or particles. In one embodiment, thebiomolecules are peptides and proteins. In some embodiments,biomolecules are produced on beads and include, but are not limited to,nucleic acids, nucleosides, nucleotides or polymers thereof (e.g. DNA orRNA). The nucleic acids, nucleosides, nucleotides or polymers thereof(e.g. DNA or RNA) can optionally be used to direct subsequent proteinsynthesis on the beads. The beads can optionally be selected, enrichedor separated based on characteristics of the biomolecules present on thebeads. In some embodiments, the biomolecules are utilized directly onthe beads for downstream assays, analyses or experiments. In otherembodiments, biomolecules are subsequently photo-transferred from thebeads to a surface, as described below.

The present invention also features methods and compositions for thephoto-transfer of compounds and/or substances from a first surface to asecond surface. In some embodiments, the photo-transferred compoundsand/or substances are deposited in highly purified and active forms.Often, the compounds and/or substances can serve as probes, targets oranalytes for bio-detection devices such as microarrays. The invention isalso directed to compositions and methods for facilitating theinteraction of compounds and/or substances. The compounds and/orsubstances can be present in a heterogeneous mixture such as blood,plasma or sera and constitute probes, targets or analytes for abio-detection device such as a microarray. Surfaces include but are notlimited to surfaces of a microarray, diagnostic devices, biochip orbio-detector. Probes, targets or analytes (so-called “features”) cancomprise compounds, substances, molecules, macromolecules and cells.Molecules and macromolecules comprise but are not limited to proteins,peptides, amino acids, amino acid analogs, nucleic acids, nucleosides,nucleotides, lipids, vesicles, detergent micelles, cells, virusparticles, fatty acids, saccharides, polysaccharides, inorganicmolecules and metals.

Direct Contact Photo-Transfer of Molecules to Surfaces

One embodiment of the invention is directed to methods for depositingcompounds and/or substances such as molecules, biomolecules,macromolecules, nanoparticles and cells from a first surface onto asecond surface. The compounds and/or substances are initially attachedto the first surface, such as present on a bead, using a photocleavablelinker or photocleavable conjugate. The first surface is then allowed todirectly contact the second surface (as distinct from another embodimentwherein the first surface is merely brought into proximity to secondsurface). The compounds and/or substances are then photo-released fromthe first surface facilitating transfer of the compounds and/orsubstances to the second surface. The first surface is then removed fromdirect contact with the second surface. This method is referred to asdirect contact photo-transfer.

In some embodiments, it is not strictly necessary that saidphoto-transferred compounds and/or substances be in physical contactwith said second surface, but in close proximity instead. Withoutlimiting the present invention to any particular mechanism, it isbelieved that it is sufficient that the compounds and/or substances bein proximity (e.g. to a distance of less than 10⁶ Angstroms, morepreferably between 0.1 and 1000 Angstroms) to said second surface. Inone embodiment, the compounds and or substances are brought intoproximity simply by bringing the surfaces into proximity (without actualcontact between the surfaces). In one embodiment, the compound isbrought into proximity via a carrier, such as a particle or bead.

In one preferred embodiment, the first surface is the surface of a beadand the second surface is chosen from substrates such as glass orplastic slides coated with nitrocellulose, PVDF or polystyrene orderivatized with aldehyde, epoxy, carboxyl, sulfhydryl or aminemoieties. In one embodiment, beads are in a solution (i.e. insuspension) before contacting the second surface and are dilutedsufficiently so that a majority of the beads deposited on the secondsurface can be individually identified. Photo-transfer of the compoundsand/or substances from the beads to the second surface, i.e. to thesubstrate, results in individually identifiable (resolved) spots on thesurface of the substrate (i.e. second surface).

In another preferred embodiment the bead is formed from agarose and iscoated with (strept)avidin or derivatives thereof. The compoundtransferred is a protein to which is attached covalently aphotocleavable biotin which binds to the (strept)avidin residing on thebead surface. The beads are deposited on epoxy coated glass slides in asolution and allowed to settle on said slides. The slide is thenilluminated with light with wavelengths longer than 300 nm (e.g. between300 nm and 400 nm, more preferably between 300 nm and 360 nm) for aperiod of time (less than one hour, more preferably less than 30minutes, still more preferably between 1 and 10 minutes, or even 1second to 1 minute). The beads are then washed from the slide with astream of water, solution or buffer.

It is to be understood that this invention is not limited by the type ofsurface that the compounds are transferred to (second surface). Such asurface may comprise glass, plastic/polymer, ceramic, or metallicmaterials either plain or additionally coated or chemicallyderivatized/conjugated with the following: antibodies;(strept)avidin/avidin or derivatives thereof such as NeutrAvidin;proteins or peptides; nucleic acids; cells; 3-dimensional matrices suchas polyacrylamide (e.g. HydroGel coated microarray substrates fromPerkinElmer Life and Analytical Sciences, Inc., Boston, Mass.) oragarose (cross-linked and non-cross-linked); membrane or film coatingssuch as nitrocellulose, polyvinylidene fluoride (PVDF) or polystyrene;polymers; chemically reactive derivitization such as activation withaldehyde, epoxy, N-hydroxy-succinimide (NHS) esters or otheramine-reactive esters such as other succinimidyl esters,tetrafluorophenyl (TFP) esters or carbonyl azides, isothiocyanate,sulfonyl chloride, cyanogen bromide, iodoacetamide or maleimidechemistries; activated surfaces such as those derivatized with amines(e.g. GAPS microarray substrates from Corning Lifesciences, Acton,Mass.) or carboxyl groups; metal ion or metal ion-chelate derivitizationsuch as nickel nitrilo-triacetic acid (Ni-NTA) or cobaltnitrilo-triacetic acid complexes/chelates; hydrophilic or hydrophobiccoatings for non-specific biomolecule absorption; gold or metal coatingssuch as those suitable for MALDI-TOF or Surface Plasmon Resonance.

Where beads or particles are used (e.g. first surface), the invention isalso not limited by the types of bead or particle used. Beads andparticles can be composed of a variety of materials including but notlimited to organic or inorganic molecules, polymer, solid-statematerials such as metals or semiconductors, biological materials, solgels, colloids, glass, magnetic materials, paramagnetic materials,electrostatic materials, electrically conducting materials, insulators,fluorescent materials, absorbing material and combinations thereof. Thebeads or particles may also vary in size, shape and density. For examplebeads may range in size from 20 nanometers to hundreds of micronsdepending on the application and spot size desired for differentapplications. The beads may also be polydisperse in regards to size,shape, material composition, optical, magnetic, electrical properties.Beads may also comprise of aggregates of smaller beads. A variety ofbead types are commercially available, including but not limited to,beads selected from agarose beads, (strept)avidin-coated beads,NeutrAvidin-coated beads, antibody-coated beads, paramagnetic beads,magnetic beads, electrostatic beads, electrically conducting beads,fluorescently labeled beads, colloidal beads, glass beads, semiconductorbeads and polymeric beads.

In addition to beads, first surface could be provided by nanoparticleshaving dimensions of 1-100 nm and more preferably 10-40 nm.Nanoparticles are a collection of atoms or molecules which are normallyin the size range of 1-100 nm and more preferable 10-50 nm. An exampleof a nanometer particle is an aggregate of several proteins or asemiconductor particle both in the size range of 1-100 nm. Compounds arebound to the surface of nanoparticles through a photocleavable linker.For example, proteins such as streptavidin can be linked throughphotocleavable linkers to the surface of nanoparticles by usingphotocleavable biotin which can be attached to the surface throughcovalent interaction and to streptavidin through non-covalentinteraction. This provides a means to release small numbers ofstreptavidin molecules in spots with a dimension approximately equal tothe projected surface areas of the nanoparticles.

Patterns of molecules can be photo-transferred onto the second surfacebased on the methods of this invention and self-assembly of thenanoparticles. The process of self-assembly of nanoparticles on asurface is well known to those working in the field of nanoparticles andfor example allows various complex patterns to be formed on a surface[Rabani, E., Reichman, D. R., Geissler, P. L., and Brus, L. E. (2003)Nature 426, 271-274]. This phenomenon thus provides a means to patternthe molecules which are photo-released from the surface ofself-assembled nanoparticles (first surface) in contact with the secondsurface.

The first surface can also comprise a flat surface such as found on aslide or surfaces with a high radius of curvature such as found on atip. Specific tips compatible with this invention include tips fromatomic force microscopes or scanning tunneling microscopes.

Regardless of what types of surfaces are employed, importantly, avariety of compounds and/or substances can be photo-transferred usingthe methods of the present invention, including but not limited tocompounds selected from the group consisting of proteins, peptides,antibodies, amino acids, amino acid analogs, drug compounds, nucleicacids, nucleosides, nucleotides, lipids, fatty acids, saccharides,polysaccharides, inorganic molecules, and metals. Photocleavage of thephotoconjugate may cause the compound or compounds to be released in amodified or unmodified form. For example, the photocleavage may leavepart of the linker attached to the compound.

Furthermore, regardless of what types of surfaces are employed, thepresent invention contemplates embodiments wherein more than one type ofaffinity reagent is used. For example, in one embodiment an antibody (afirst affinity reagent) is covalently conjugated to a photocleavablebiotin (a second affinity reagent). The antibody is directed against oneor more epitopes which are part of a protein. The protein then becomesbound to (strept)avidin coated agarose beads (a third affinity reagent)through the interaction of biotin on the antibody with (strept)avidin onthe beads and interaction of the antibody with the protein.

It is to be understood that either a single homogeneous compound orsubstance, or a mixture of different compounds and/or substances can bebound to the first surface through photocleavable linkers. In the casewhere first surface comprises beads or nanoparticles, a mixture of beadsor nanoparticles, each containing different compounds and/or substances,can be used to photo-transfer the compound(s) and/or substance(s) ontothe second surface. For example, in the case of beads, each spotresulting from photo-transfer may thus contain a mixture of compoundsand/or substances or each spot may contain one compound and/or substancewhich is different from another spot.

In one preferred embodiment, the compound to be deposited on the secondsurface is a target molecule present in a biological fluid such as wholeblood or sera. An antibody directed against the target molecule is boundthrough a photocleavable conjugate to a bead (first surface). Aftercontacting the biological fluid, the beads are separated from thebiological fluid and allowed to directly contact the second surface. Thebeads are then illuminated at preferred wavelengths of light whichcauses photo-transfer of the antibody-target molecule complex to thesecond surface.

Other useful biological fluids include but are not limited to saliva,cerebrospinal fluid, synovial fluid, urine and sweat. Additionalbiological samples include but are not limited to stool samples andtumors (e.g. biopsies, tumor cell lines, primary cultures, lysates,etc.). The photo-conjugate can comprise of a photocleavable biotincovalently bound to an antibody which is directed against an antigen.The bead (first surface) comprises a (strept)avidin or avidin orderivatives thereof such as NeutrAvidin, coated onto a porous polymermatrix such as agarose, Sepharose or polyacrylamide. The photo-conjugateis linked to the bead through a biotin-(strept)avidin interaction. Afterthe beads are washed away from the second surface, a labeled antibodywhich selectively interacts with the antigen is added for detectionpurposes.

In comparison to a conventional sandwich immunoassay, well known in thefield for detection of analytes, the present invention avoids thepotential transfer of non-specifically bound materials present in theblood, plasma, sera or biological fluid for example, from the firstsurface to the second surface, due to selective and gentle release ofthe target analyte from the first surface using photocleavage.

In another preferred embodiment, the compound to be deposited on thesecond surface is a target antibody present in a biological fluid suchas whole blood or sera. An antigen for the target antibody (e.g. aspecific allergen which interacts with a target specific IgE) is boundthrough a photocleavable linker to a bead (first surface). Aftercontacting the biological fluid to allow the antibody-antigeninteraction, the beads are separated from the biological fluid andallowed to directly contact the second surface. The beads are thenilluminated at preferred wavelengths of light under conditions such thatsaid antigen-antibody complexes are photocleaved from said beads andtransferred to said second surface.

In an additional preferred embodiment, a nascent protein is synthesizedin a cellular or cell-free transcription/translation system, whereby thenascent protein contains one or more affinity markers. Beads coated withan affinity agent which selectively binds to the affinity marker areallowed to contact the cellular or cell-free transcription/translationsystem. The beads are then separated from the transcription/translationsystem and allowed to directly contact the second surface. The beads arethen illuminated under conditions such that said nascent proteins arephotocleaved from said beads and transferred to said second surface.

In one preferred embodiment, compounds and/or substances (e.g. theproteins) are transferred from a multiarray probe device such as presenton an AFM tip array described previously (Green, J-B D., Novoradovsky, Aet al., Phys. Rev. Letts 74, 1999, 1489) to a second surface. Compoundsand/or substances are bound to the tips through a photocleavable linker.The tips are allowed to contact a second surface and then illuminatedwith light to facilitate the transfer of said compounds and/orsubstances to the second surface. Since the tips are nanometer scale orless, only small nanometer scale spots comprising the compounds and/orsubstances (e.g. protein) will be transferred to the second surface.

In another preferred embodiment, a suspension of nanoparticles isspotted onto the second surface using a conventional robotic spotter(microarray printing device), or randomly dispersed on a second surface.Different compounds and/or substances are linked to differentnanoparticles through photocleavable linkers. The nanoparticles areilluminated with radiation under conditions such that the compoundsand/or substances are photocleaved from said nanometer particles andtransferred to said second surface.

As described in U.S. Pat. No. 5,643,722, which is specificallyincorporated by reference, and variations thereof described in U.S. Pat.No. 6,306,628, which is also specifically incorporated by reference,affinity markers containing photocleavable bonds can be incorporatedinto nascent proteins during their cell-free synthesis. In one example,specially prepared tRNAs are used to incorporate a photocleavable biotinin place of one or more normal residues in the proteins amino acidsequence. Such photocleavable linkers can also be incorporatedspecifically at the N-terminal end of the protein by using initiatorsuppressor tRNA. This provides a means to capture these nascent proteinsselectively from the rest of the protein synthesis system, onto thefirst surface, followed by protein transfer to the second surface usingthe methods described in this invention.

Affinity markers in the form of epitopes can also be incorporated into anascent proteins by designing the message or DNA coding for the nascentprotein to have a nucleic acid sequence corresponding to the particularepitope. This can be accomplished by using primers that incorporate thedesired nucleic acid sequence into the DNA coding for the nascentprotein using the polymerase chain reaction (PCR). A variety of epitopetag sequences can be utilized in the methods of the present invention,including His₆ (or other polyhistidine tags), c-myc, a p53-tag (derivedfrom the P53 sequence), HSV, HA, FLAG, VSV-G, Fil-16 (filamin derived)and StrepTag.

In addition to proteins, methods and compositions of this invention aredirected to depositing nucleic acid molecules or macromoleculescontaining nucleic acids (heretofore “nucleic acid” is meant to includeall complexes containing nucleic acids) onto a second surface. Thenucleic acid molecules are initially attached to a different surface(first surface), such as present on a bead, with the attachment being bya photocleavable linker or conjugate. The first surface is then allowedto directly contact the second surface. The nucleic acid molecules arethen photo-released from the first surface facilitating transfer of thenucleic acid to the second surface. The first surface is then removedfrom direct contact with the second surface.

In one preferred embodiment, a nucleic acid molecule is synthesized witha photocleavable affinity tag. Beads coated with an affinity agent whichbinds to the affinity marker are allowed to contact a solutioncontaining the nucleic acid molecules or nucleic acid complexes. Beadsare then separated from the solution and allowed to directly contact thesecond surface. The beads are then illuminated at preferred wavelengthwhich causes photo-transfer of the nucleic acids molecules to the secondsurface. It will be understood by those skilled in the art of nucleicacid chemistry there exists a number of methods to incorporatephotocleavable tags into nucleic acid molecules during or after theirsynthesis, including methods based on enzymatic incorporation orchemical synthesis.

In one example, photocleavable biotins are incorporated into nucleicacids or nucleic acid complexes. The incorporation of photocleavablebiotin and other photocleavable affinity markers are described in U.S.Pat. No. 5,643,722 which is specifically incorporated by reference, andvariations thereof described in U.S. Pat. Nos. 5,986,076 and 6,057,096,which are also specifically incorporated by reference.

As described in U.S. Pat. No. 6,057,096, the isolation of nucleic acidsis based on three basic steps. First, a photocleavable biotin derivativeis attached to a nucleic acid molecule by enzymatic or chemical meansor, alternatively, by incorporation of a photocleavable biotinnucleotide into a nucleic acid by enzymatic or chemical means. Thechoice of a particular photocleavable biotin depends on which moleculargroups are to be derivatized on the nucleic acid. For example,attachment of photocleavable biotin to a nucleic acid can beaccomplished by forming a covalent bond with the aromatic amine, sugarhydroxyls or phosphate groups. Photocleavable biotin can also beincorporated into oligonucleotides through chemical or enzymatic means.

In some embodiments, there is no need for external printing methods suchas performed by conventional robotic printing. Instead, a spot is formedon the second surface in the immediate vicinity of where the beads ornanoparticles (first surface) contact the second surface. Furthermore,the shape (i.e. morphology) of the spot is directly related to the sizeand shape of the contacting surface (first surface) such as from a beador nanoparticles.

As in the case of conventional printing, the interaction between thephoto-released molecule and the second surface determines in part howwell the molecule will adhere to the second. For example, proteins willform covalent linkage with some specific surfaces which have present attheir surface specifically activated (i.e. reactive) molecules. Forexample, commercially available glass slides, such as those derivatizedwith epoxy or aldehyde moieties, have particular chemical groupsallowing particular interactions. A second example involves proteinswhich interact strongly with nitrocellulose, PVDF or polystyrenesurfaces, mainly through hydrophobic interactions. A third example isthe use of a secondary antibody bound to a surface, which is chosen tointeract selectively with the primary antibody involved in thephotocleavable conjugate. An fourth example involves hydrated matrixcoated slides which bind proteins (e.g. polyacrylamide gels or HydroGelcoated microarray substrates; PerkinElmer Life and Analytical Sciences,Inc., Boston, Mass.). A fifth example involves surfaces (e.g. slides,chips, etc.) with charged chemical groups such as amines or carboxyls,which can non-covalently bind proteins through ionic interactions, orcan be covalently linked to proteins with the aid of chemically reactivecross-linkers.

In one preferred embodiment, the second surface comprises a MALDIsubstrate which is normally coated with gold. The gold surface isactivated by chemically incorporating reactive groups which interactwith different types of molecules including hydrophobic, hydrophilic andmolecules containing specific chemical groups [Koopmann & Blackburn.(2003) Rapid Commun Mass Spectrom 17, 455-462; Zhang & Orlando. (1999)Anal Chem 71, 4753-4757; Neubert et al. (2002) Anal Chem 74, 3677-3683;Kiernan et al. (2002) Clin Chem 48, 947-949; Darder et al. (1999) AnalChem 71, 5530-5537].

Conventional microarray printers (e.g. spotters) can be used to depositone or more beads (first surface) at specific positions on a secondsurface. In some cases it is desirable to deposit a single bead perspot. This can be achieved, in one embodiment, by diluting the beadssolution so that each liquid spot deposited (e.g. by the robot) has atmost one bead. The density of beads deposited per spot can be controlledby a number of factors well known in the field. For example, thediameter of capillary fibers used in the printing process can becontrolled so that the inner diameter of the fiber is restricted to asingle file of beads. Alternatively, the drop size in the case of inkjet printing technology can be used to control the number of beadsdeposited per spot. Alternatively, beads can be deposited on a surfacecomprising wells, wherein said wells are dimensioned to permit one beador particle, and not more than one bead or particle, to fit or at leastpartially fit or settle.

The present invention also contemplates methods which do not requiremechanical microarray printers. For example, beads (first surface) whichcontain photocleavable conjugates that link various molecules can beallowed to contact the second surface by introducing all of the beadstogether in solution form, i.e. in suspension, which contacts the secondsurface. The bead deposition in this case will cause a random orsemi-random pattern. In order to control the average 2-dimensionaldensity of beads on the second surface, the solution of beads whichcontacts the surface can be diluted to a desired concentration. Othermethods of introducing the beads without the use of a mechanicalmicroarray printer include spraying the beads onto the surface.

Alternatively, beads or nanoparticles (first surface) can be guided tospecific positions on the second surface without the use of conventionalmechanical microarray printers, using preexisting features on the secondsurface. For example, interaction of beads or nanoparticles withpreformed elements on the second surface include but are not limited tomechanical (e.g. etched wells, dimples or holes), electrostatic,magnetic, surface tension, capillarity, molecular interactions, covalentinteracts, DNA hybridization and protein-protein interactions.

A variety of approaches can be used to modify a second surface to guidebeads (first surface) to specific positions. One example of preformedfeatures on a second surface which can be used to guide beads tospecific positions is based on utilization of small etched pits whichhold the beads. Such a mechanism is used for example in the case ofIllumina's (Illumina Incorporated; San Diego, Calif.) coded bead arraytechnology [Gunderson, K. L. et al., (2004) Genome Res 14, 870-877].

Regardless of the methods, compounds, substances and/or surfaces used inthis invention, it is not intended that the present invention be limitedto particular photocleavable linkers used in the photo-transfer process.There are a variety of known photocleavable linkers. Preferred comprisea 2-nitrobenzyl moiety. U.S. Pat. No. 5,643,722 describes a variety ofsuch linkers and is hereby incorporated by reference.

Identifying Molecules Deposited by Direct Contact Photo-Transfer ofCoding Agents

Another embodiment of the invention is directed to methods fordetermining the identity of compounds deposited in a plurality of spotson a second surface using the methods of direct contact photo-transfer.A plurality of beads or nanoparticles are prepared with coding agentssuch that different compounds or mixture of compounds are linked usingphotocleavable conjugates to different beads containing different codingagents. The coding agents allow beads (and the photo-transferredcompound(s)) to be uniquely identified on the basis of unique spectral,mechanical, magnetic or electrical properties which identifies on codingagent from another. Following methods of this invention for preparingthese beads, the beads are allowed to directly contact the secondsurface. In one embodiment, the coding properties of each bead are thenrecorded as a function of position on the bead on the second surface.The beads are then illuminated causing photo-transfer of the compoundsfrom each bead to the second surface. The beads are then removed fromthe surface. Later the information recorded about bead coding as afunction of position is used to identify the compound or compoundsdeposited in each spot.

It is not intended that the present invention be limited to the natureof the particular coding method. A variety of methods are known forcoding beads some of which are commercially available. In general,several categories of coding options can be used in the context ofdirect contact photo-transfer, including but not limited to:

Spectral Coding: Agents having unique and distinguishable spectralproperties can be used for decoding following contact photo-transfer.One embodiment for spectral coding utilizes fluorescent quantum dotnanocrystals. Based on published reports, such as by Han et al. [Han etal. (2001) Nat Biotechnol 19, 631-635], highly fluorescent quantum dotnanocrystals can be used for spectral bar coding on beads. As many as40,000 distinct codes can be created by adjusting the ratio of theintensities of different quantum dot species having differentfluorescence emissions (“colors”). For example, nearly 1,000 distinctcodes can be achieved using 3 colors of quantum dot nanocrystals at 10different intensity levels (10³−1=999 codes). Quantum dot nanocrystalcodes can be photocleavably attached to a first surface (e.g. bead) tofacilitate contact photo-transfer to a receiving surface (secondsurface). In one embodiment, to facilitate photocleavable attachment tothe first surface (e.g. bead), protein or amine functionalized quantumdot nanocrystals (e.g. from Quantum Dot Corporation, Hayward, Calif.)can be labeled with AmberGen's amine reactive photocleavable biotin(PC-biotin) reagent (AmberGen Incorporated, Waltham, Mass.) [Olejnik etal. (1995) Proceedings of the National Academy of Science (USA) 92,7590-7594; Pandori et al. (2002) Chem Biol 9, 567-573].

DNA Coding: DNA decoding schemes have been previously reported forbead-based fiber-optic microarray devices used in detection ofsingle-nucleotide polymorphisms (SNPs) (Illumina Inc., San Diego,Calif.) [Gunderson et al. (2004) Genome Res 14, 870-877]. In thisapproach, each bead is encoded with a unique DNA sequence (code) whichcan be read by hybridization probes consisting of fluorescently labeledcomplementary oligonucleotides (decoders). A highly efficient algorithmhas been developed which allows thousands of different sequences to beidentified with just a few color probes and several cycles ofhybridization. For example, 1,520 unique DNA sequences have been decodedusing 3 colors (blank, red and green) and 7 sequential hybridizationsteps (with each hybridization step containing 1,520 decoder probes,each carrying one of the 3 possible colors; color on decoder probes ismodulated for each sequential hybridization step to achieve all 1,520codes).

DNA codes can be photocleavably attached to a first surface (e.g. bead)to facilitate contact photo-transfer to a receiving surface (secondsurface). DNA coding elements can be manually attached to the firstsurface or generated via solid-phase bridge PCR for example. In oneembodiment, a photocleavable amine phosphoramidite reagent, soldcommercially by Glen Research (Sterling, Va.; http://www.glenres.com),can be used to photocleavably attach DNA codes. This phosphoramiditewill generate a photocleavable 5′ amine modified oligonucleotide, whichcan then be attached to amine-reactive beads or attached to beads viaamine-based cross-linking chemistries (e.g. carbodiimide based couplingto carboxyl functionalized beads).

Protein/Peptide Coding: Proteins, polypeptides or peptides which can bedistinguished based on certain characteristics can also serve as codingagents following contact photo-transfer. In one embodiment,peptide/protein codes of unique and distinguishable masses are contactphoto-transferred to a receiving surface (second surface) andsubsequently detected using matrix-assisted laser desorption/ionizationmass spectrometry (MALDI-MS). A similar application of photocleavablemass tags for multiplexed assays has been previously reported byAmberGen [Olejnik et al. (1999) Nucleic Acids Res 27, 4626-4631; Hahneret al. (1999) Biomol Eng 16, 127-133]. Only relatively small peptides(e.g. 10 amino acids) are required to create tens of thousands of masstags with unique masses that can be easily distinguished with a highresolution mass spectrometer.

Protein/Peptide codes can be photocleavably attached to a first surface(e.g. bead) to facilitate contact photo-transfer to a receiving surface(second surface). Protein/Peptide coding elements can be manuallyattached to the first surface or generated, for example, via cell-freeprotein synthesis from DNA on the first surface, whereby in situprotein/peptide capture [Ramachandran et al. (2004) Science 305, 86-90;Nord et al. (2003) J Biotechnol 106, 1-13] is used to isolate theprotein/peptide code onto the same bead from which it was produced;using for example, a photocleavably linked antibody for capture of theprotein/peptide codes (proteins/peptides can be comprised of a commonepitope tag for antibody capture and a variable region for coding).

One specific example of spectral coding involves the use of Qbeadsoffered by Quantum Dot Corporation (Hayward, Calif.). Qbeads coding isbased on spectral bar-coding. In the case of Qbeads, microspheres aredyed with Qdot® nanocrystals (referred to a as quantum dots) which aresmall crystals ranging in size from 10-30 nm. Different nanocrystalshave different distinct fluorescent excitation spectra, thus allowingcodes to be created on the basis of the different types of nanocrystalsand their relative ratio attached to a particular Qbead. Whenilluminated with UV or visible light, these encoded spheres emit withthe characteristics of the underlying quantum dots. Differentpopulations of beads can be encoded with different ratios and differentcombinations of quantum dots colors. Beads can then be mixed but theirindividual identity can be determined by measuring the fluorescentproperties of the beads. This can be performed for example using afluorescence microscope or microarray scanner with multicolor capabilitysuch as the ArrayWoRx scanner manufactured by Applied Precision Inc. Inprinciple, the number of quantum dots available with different spectralproperties can allow as many as a million different unique spectralcodes to be created enabling multiplexed read-out of large numbers ofbeads.

Previously, Qbeads have been used for a number of biotechnologicalapplications including SNP genotyping (Xu et al. (2003) “Multiplexed SNPgenotyping using the Qbead™ system: a quantum dot-encodedmicrosphere-based assay,” Nucleic Acids Res. 31 (8):e43). A variety ofmethods of probing quantum dots can be used for the decoding process andhave been described in the literature (Alivisatos, A. P. (2004) “The Useof Nanocrystals in Biological Detection,” Nature Biotech. 22:47-52).

It is to be understood that it is not intended that the presentinvention be limited to coding agents that are quantum dots and the useof fluorescent spectral properties. For example, beads can be codedbased on their infrared, Raman or resonance Raman spectrum by adding avariety of compounds with easily identifiable vibrational spectralfeatures (Fenniri, H., Chun, S., Ding, L., Zyrianov, Y., and Hallenga,K. (2003) J Am Chem Soc 125, 10546-10560). Beads can also be coded usinga combination of molecules with unique absorption spectra in the visibleor UV spectral range. An additional spectral property useful for codingbeads is the nuclear magnetic resonance spectrum of one or morecompounds. Beads can also be coded by attaching a unique polymer whichcan be sequenced. In one embodiment, unique sequences of nucleic acidsare attached to beads, removed and sequenced or alternatively removed,amplified using polymerase chain reaction and sequenced. In yet anotherapproach, beads can be coded by attaching molecules with uniquemolecular masses and detected using mass spectrometry.

Coding may also be provided by the intrinsic properties of the compoundto be photo-transferred to second surface. For example, the compound canbe identified on the basis of a unique molecular mass by using massspectrometry. Compounds to be photo-transferred may also have uniquespectral characteristics including V, visible, infrared absorption orfluorescent emission spectra and NMR spectrum. In one embodiment, uniquecombinations of different species of green fluorescent protein whichhave different emission and excitation spectra are used for coding.

Another preferred embodiment of the invention is directed to methods fordetermining the identity of compounds deposited in a plurality of spotson a surface by incorporating on the bead coding agents which arephoto-transferred along with the compounds to the second surface. Aplurality of coded beads are prepared such that different compounds ormixture of compounds are linked using photocleavable conjugates todifferent beads with unique coding. In addition, the coding agents areattached to the beads using photocleavable conjugates. The beads arethen isolated and allowed to directly contact second surface. The beadsare then illuminating with preferred wavelengths causing photo-releaseand deposition of the compounds and the coding agents. The beads arethen removed from the second surface. The identity of the compoundsdeposited in each spot is then determined by measuring some property ofthe photo-transferred coding agents.

A variety of methods and compositions are contemplated in this inventionfor producing photo-transferable coding agents which are used todetermine the identity of compounds photo-transferred from beads ontosecond surface. In one example, these coding agents comprisenanocrystals with distinct spectral properties such as Qdot®. Differentnanocrystals have different distinct fluorescent excitation spectra,thus allowing codes to be created on the basis of the different types ofnanocrystals and their relative ratio.

In one preferred embodiment, both the compounds to be photo-transferredalong quantum dots are linked through photocleavable conjugates tobeads. The beads are then allowed to contact second surface. The beadsare then illuminated with preferred wavelengths to photo-release boththe compounds and the quantum dots. The beads are then removed bywashing leaving behind spots containing both the photodepositedcompounds and quantum dots which serve as coding agents allowingidentification of the compounds.

For example, Quantum Dot Corporation offers a Qdot® antibody conjugationkits for 565, 605, 655 and 705 nm fluorescent emitting nanoocrystals.Quantum Dot Corporation introduced this kit to allow researchers toconjugate their antibody of choice to nanocrystals. However, a similarprocedure can be used to create nanocrystals which containphotocleavable linkers to beads. In particular Qdots nanocrystalscontain a number of amine groups on their surfaces. In the prescribedprocedures included in the kit, the amine groups are converted tothiol-reactive maleimide groups for the purpose of linking antibodies.However, these amine groups are also reactive against specific PC-linkerreagents such as NHS-PC-biotin and other photocleavable affinity markerswhich are described in U.S. Pat. No. 5,986,076 which is specificallyincorporated by reference, and variations thereof described in U.S. Pat.No. 6,057,096, which is also specifically incorporated by reference.

In a typical procedure designed to create photocleavable nanocrystalswhich can be linked to a variety of beads and surfaces, the Qdotsdescribed above are treated with NHS-PC-biotin. After conjugation,unreacted NHS-PC-biotin is removed. The purified nanocrystal-PC-biotinconjugate is then contacted with beads or a surface to whichstreptavidin or derivatives are bound in order to link the nanocrystalsto the beads. The present invention specifically contemplates, ascompositions of matter, nanocrystal photocleavable biotin conjugates aswell as beads comprising nanocrystal-photocleavable-biotin conjugates.

It is understood that this invention is not limited to the nature of thenanocrystals, photolinker or bead. For example, a variety of methodshave been reported for coating nanocrystals with surfaces which can bemade specifically reactive thereby allowing photocleavable linkers to beconjugated (Lingerfelt, B. M., Mattoussi, H., Goldman, E. R., Mauro, J.M., and Anderson, G. P. (2003) Anal Chem 75, 4043-4049).

Polymeric microspheres or beads can be prepared from a variety ofdifferent polymers, including but not limited to polystyrene,cross-linked polystyrene, polyacrylic, polylactic acid, polyglycolicacid, poly(lactide coglycolide), polyanhydrides, poly(methylmethacrylate), poly(ethylene-co-vinyl acetate), polysiloxanes, polymericsilica, latexes, dextran polymers and epoxies. The materials have avariety of different properties with regard to swelling and porosity,which are well understood in the art. Preferably, the beads are in thesize range of approximately 10 nm to 1 mm (and more preferably in thesize range between approximately 50 nm and 500 nm), and can bemanipulated using normal solution techniques when suspended in asolution.

A plurality of such beads or mixtures of different bead populations canbe immobilized on a planar surface such that they are regularly spacedin a chosen geometry using any suitable method. For example, beads canbe immobilized by micromachining wells in which beads can be entrappedinto the surface, or by patterned activation of polymers on the surfaceusing light activation to cross-link single beads at particularlocations. Suitable wells can be created by ablating circles in a layerof parylene deposited on a glass surface using a focused laser. In oneembodiment, the well dimensions are chosen such that a single bead canbe captured per well. For example, 7 micron wells can be readily usedfor analysis of beads about 4 microns to about 6 microns in diameter.This can be performed (if desired) on the end of an optical fiber. Onthe other hand, the well dimensions in other embodiments may be chosensuch that two or more beads can be captured per well. Whether the welldimensions accommodate one bead or a plurality of beads, it is preferredthat the wells not be so deep that the beads remain trapped when alateral flow of fluid passes across the surface. On the other hand, insome embodiments, it may be desirable to dimension the wells so as tocause the beads to remain trapped when a lateral flow of fluid passesacross the surface.

The present invention contemplates beads comprising coding agents to asto create spectrally encoded microspheres. Microspheres can bespectrally encoded through incorporation of semiconductor nanocrystals(or SCNCs). The desired fluorescence characteristics of the microspheresmay be obtained by mixing SCNCs of different sizes and/or compositionsin a fixed amount and ratio to obtain the desired spectrum, which can bedetermined prior to association with the microspheres. Subsequenttreatment of the microspheres (through for example covalent attachment,co-polymerization, or passive absorption or adsorption) with thestaining solution results in a material having the designed fluorescencecharacteristics.

A number of SCNC solutions can be prepared, each having a distinctdistribution of sizes and compositions, to achieve the desiredfluorescence characteristics. These solutions may be mixed in fixedproportions to arrive at a spectrum having the predetermined ratios andintensities of emission from the distinct SCNCs suspended in thatsolution. Upon exposure of this solution to a light source, the emissionspectrum can be measured by techniques that are well established in theart. If the spectrum is not the desired spectrum, then more of the SCNCsolution needed to achieve the desired spectrum can be added and thesolution “titrated” to have the correct emission spectrum. Thesesolutions may be colloidal solutions of SCNCs dispersed in a solvent, orthey may be pre-polymeric colloidal solutions, which can be polymerizedto form a matrix with SCNCs contained within.

The SCNCs can be attached to the beads by covalent attachment as well asby entrapment in swelled beads, or can be coupled to one member of abinding pair the other member of which is attached to the beads. Forinstance, SCNCs are prepared by a number of techniques that result inreactive groups on the surface of the SCNC. See, e.g., Bruchez et al.(1998) Science 281:2013-2016, Chan et al. (1998) Science 281:2016-2018,Colvin et al. (1992) J. Am. Chem. Soc. 114:5221-5230, Katari et al.(1994) J. Phys. Chem. 98:4109-4117, Steigerwald et al. (1987) J. Am.Chem. Soc. 110:3046.

The reactive groups present on the surface of the SCNCs can be coupledto reactive groups present on a surface of the material. For example,SCNCs which have carboxylate groups present on their surface can becoupled to beads with amine groups using a carbodiimide activation step.Any cross-linking method that links a SCNC to a bead and does notadversely affect the properties of the SCNC or the bead can be used. Ina cross-linking approach, the relative amounts of the different SCNCscan be used to control the relative intensities, while the absoluteintensities can be controlled by adjusting the reaction time to controlthe number of reacted sites in total. After the beads are crosslinked tothe SCNCs, the beads are optionally rinsed to wash away unreacted SCNCs.

In some embodiments, a sufficient amount of fluorophore must be used toencode the beads so that the intensity of the emission from thefluorophores can be detected by the detection system used and thedifferent intensity levels must be distinguishable, where intensity isused in the coding scheme but the fluorescence emission from the SCNCsor other fluorophores used to encode the beads must not be so intense toas to saturate the detector used in the decoding scheme.

The beads or other substrate to which one or more different knowncapture probes are conjugated can be encoded to allow rapid analysis ofbead, and thus capture probe, identity, as well as allowingmultiplexing. The coding scheme preferably employs one or more differentSCNCs, although a variety of additional agents, including chromophores,fluorophores and dyes, and combinations thereof can be usedalternatively or in combination with SCNCs. For organic dyes, differentdyes that have distinguishable fluorescence characteristics can be used.Different SCNC populations having the same peak emission wavelength butdifferent peak widths can be used to create different codes ifsufficient spectral data can be gathered to allow the populations to bedistinguished. Such different populations can also be mixed to createintermediate linewidths and hence more unique codes.

The number of SCNCs used to encode a single bead or substrate locale canbe selected based on the particular application. Single SCNCs can bedetected; however, a plurality of SCNCs from a given population ispreferably incorporated in a single bead to provide a stronger, morecontinuous emission signal from each bead and thus allow shorteranalysis time.

Different SCNC populations can be prepared with peak wavelengthsseparated by approximately 1 nm, and the peak wavelength of anindividual SCNC can be readily determined with 1 nm accuracy. In thecase of a single-peak spectral code, each wavelength is a differentcode. For example, CdSe SCNCs have a range of emission wavelengths ofapproximately 490-640 nm and thus can be used to generate about 150single-peak codes at 1 nm resolution.

A spectral coding system that uses only highly separated spectral peakshaving minimal spectral overlap and does not require stringent intensityregulation within the peaks allows for approximately 100,000 to10,000,000 or more unique codes in different schemes.

A binary coding scheme combining a first SCNC population having anemission wavelength within a 490-565 nm channel and a second SCNCpopulation within a 575-650 nm channel produces 3000 valid codes using1-nm resolved SCNC populations if a minimum peak separation of 75 nm isused. The system can be expanded to include many peaks, the onlyrequirement being that the minimum separation between peak wavelengthsin valid codes is sufficient to allow their resolution by the detectionmethods used in that application.

A binary code using a spectral bandwidth of 300 nm, a coding-peakresolution, i.e., the minimum step size for a peak within a singlechannel, of 4 nm, a minimum interpeak spacing of 50 nm, and a maximum of6 peaks in each code results in approximately 200,000 different codes.This assumes a purely binary code, in which the peak within each channelis either “on” or “off.” By adding a second “on” intensity, i.e.,wherein intensity is 0, 1 or 2, the number of potential codes increasesto approximately 5 million. If the coding-peak resolution is reduced to1 nm, the number of codes increases to approximately 1×10¹⁰.

Valid codes within a given coding scheme can be identified using analgorithm. Potential codes are represented as a binary code, with thenumber of digits in the code corresponding to the total number ofdifferent SCNC populations having different peak wavelengths used forthe coding scheme. For example, a 16-bit code could represent 16different SCNC populations having peak emission wavelengths from 500 nmthrough 575 nm, at 5 nm spacing. A binary code 1000 0000 0000 0001 inthis scheme represents the presence of the 500 nm and 575 nm peaks. Eachof these 16-bit numbers can be evaluated for validity, depending on thespacing that is required between adjacent peaks; for example, 0010 01000000 0000 is a valid code if peaks spaced by 15 nm or greater can beresolved, but is not valid if the minimum spacing between adjacent peaksmust be 20 nm. Using a 16-bit code with 500 to 575 nm range and 5 nmspacing between peaks, the different number of possible valid codes fordifferent minimum spectral spacings between adjacent peaks is shown inTable 1.

TABLE 1 The number of unique codes with a binary 16-bit system. SpectralSeparation 5 nm 10 nm 15 nm 20 nm 25 nm 30 nm Number of 65535 2583 594249 139 91 unique codesIf different distinguishable intensities are used, then the number ofvalid codes dramatically increases. For example, using the 16-bit codeabove, with 15 nm minimum spacing between adjacent peaks in a code,7,372 different valid codes are possible if two intensities, i.e., aternary system, are used for each peak, and 38,154 different valid codesare possible for a quaternary system, i.e., wherein three “on”intensities can be distinguished.

Codes utilizing intensities require either precise matching ofexcitation sources or incorporation of an internal intensity standardinto the beads due to the variation in extinction coefficient exhibitedby individual SCNCs when excited by different wavelengths.

In some embodiments, it is preferred that the light source used for theencoding procedure be as similar as possible (preferably of the samewavelength and intensity) to the light source that will be used fordecoding. The light source may be related in a quantitative manner, sothat the emission spectrum of the final material may be deduced from thespectrum of the staining solution.

An example of an imaging system for automated detection of nanocrystalsfor use with the present methods comprises an excitation source, amonochromator (or any device capable of spectrally resolving the image,or a set of narrow band filters) and a detector array. The excitationsource can comprise blue or UV wavelengths shorter than the emissionwavelength(s) to be detected. This may be: a broadband UV light source,such as a deuterium lamp with a filter in front; the output of a whitelight source such as a xenon lamp or a deuterium lamp after passingthrough a monochromator to extract out the desired wavelengths; or anyof a number of continuous wave (cw) gas lasers, including but notlimited to any of the Argon Ion laser lines (457, 488, 514, etc. nm) ora HeCd laser; solid state diode lasers in the blue such as GaN and GaAs(doubled) based lasers or the doubled or tripled output of YAG or YLFbased lasers; or any of the pulsed lasers with output in the blue.

The emitted light can be detected with a device that provides spectralinformation for the substrate, e.g., grating spectrometer, prismspectrometer, imaging spectrometer, or the like, or use of interference(bandpass) filters. Using a two-dimensional area imager such as a CCDcamera, many objects may be imaged simultaneously. Spectral informationcan be generated by collecting more than one image via differentbandpass, longpass, or shortpass filters (interference filters, orelectronically tunable filters are appropriate). More than one imagermay be used to gather data simultaneously through dedicated filters, orthe filter may be changed in front of a single imager. Imaging basedsystems, like the Biometric Imaging system, scan a surface to findfluorescent signals.

A scanning system can be used in which the sample to be analyzed isscanned with respect to a microscope objective. The luminescence is putthrough a single monochromator or a grating or prism to spectrallyresolve the colors. The detector is a diode array that then records thecolors that are emitted at a particular spatial position. The softwarethen recreates the scanned image.

When imaging samples labeled with multiple fluorophores, it is desirableto resolve spectrally the fluorescence from each discrete region withinthe sample. Such samples can arise, for example, from multiple types ofSCNCs (and/or other fluorophores) being used to encode beads, from asingle type of SCNC being used to encode beads but bound to a moleculelabeled with a different fluorophore, or from multiple molecules labeledwith different types of fluorophores bound at a single location. Manytechniques have been developed to solve this problem, including Fouriertransform spectral imaging (Malik et al. (1996) J. Microsc. 182:133;Brenan et al. (1994) Appl. Opt. 33:7520) and Hadamard transform spectralimaging, or simply scanning a slit or point across the sample surface(Colarusso et al. (1998) Appl. Spectrosc. 52: 106A), all of which arecapable of generating spectral and spatial information across atwo-dimensional region of a sample.

One-dimensional spectral imaging can easily be achieved by projecting afluorescent image onto the entrance slit of a linear spectrometer. Inthis configuration, spatial information is retained along the y-axis,while spectral information is dispersed along the x-axis (Empedocles etal. (1996) Phys. Rev. Lett. 77(18):3873). The entrance slit restrictsthe spatial position of the light entering the spectrometer, definingthe calibration for each spectrum. The width of the entrance slit, inpart, defines the spectral resolution of the system.

Two-dimensional images can be obtained by eliminating the entrance slitand allowing the discrete images from individual points to define thespatial position of the light entering the spectrometer. In this case,the spectral resolution of the system is defined, in part, by the sizeof the discrete images. Since the spatial position of the light fromeach point varies across the x-axis, however, the calibration for eachspectrum will be different, resulting in an error in the absolute energyvalues. Splitting the original image and passing one half through adispersive grating to create a separate image and spectra can eliminatethis calibration error. With appropriate alignment, a correlation can bemade between the spatial position and the absolute spectral energy.

To avoid ambiguity between images that fall along the same horizontalline, a second beam-splitter can be added, with a second dispersiveelement oriented at 90 degrees to the original. By dispersing the imagealong two orthogonal directions, it is possible to unambiguouslydistinguish the spectra from each discrete point within the image. Thespectral dispersion can be performed using gratings, for exampleholographic transmission gratings or standard reflection gratings. Forexample, using holographic transmission gratings, the original image issplit into 2 (or 3) images at ratios that provide more light to thespectrally dispersed images, which have several sources of light loss,than the direct image. This method can be used to spectrally image asample containing discrete point signals, for example in high throughputscreening of discrete spectral images such as single molecules orensembles of molecules immobilized on a substrate, and for highlyparallel reading of spectrally encoded beads. The images are thenprojected onto a detector and the signals are recombined to produce animage that contains information about the amount of light within eachband-pass.

Alternatively, techniques for calibrating point spectra within atwo-dimensional image are unnecessary if an internal wavelengthreference (the “reference channel”) is included within the spectrallyencoded material. The reference channel is preferably either the longestor shortest wavelength emitting fluorophore in the code. The knownemission wavelength of the reference channel allows determination of theemission wavelengths of the fluorophores in the dispersed spectral codeimage. In addition to wavelength calibration, the reference channel canserve as an intensity calibration where coding schemes with multipleintensities at single emission wavelengths are used. Additionally, afixed intensity of the reference channel can also be used as an internalcalibration standard for the quantity of label bound to the surface ofeach bead.

In one system for reading spectrally encoded beads or materials, aconfocal excitation source is scanned across the surface of a sample.When the source passes over an encoded bead, the fluorescence spectrumis acquired. By raster-scanning the point-excitation source over thesample, all of the beads within a sample can be read sequentially.

Optical tweezers can optionally be used to “sweep” spectrally encodedbeads or any other type of bead into an ordered array as the beads areread. The “tweezers” can either be an infrared laser that does notexcite any fluorophores within the beads, or a red laser thatsimultaneously traps the beads and also excites the fluorophores.Optical tweezers can be focused to a tight spot in order to hold amicron-size bead at the center of this spot by “light pressure.”

Optical tweezers can be used to hold spectrally encoded beads and toorder them for reading. The tweezers can be focused near the bottom of awell located at the center of the detection region of a point-scanningreader, which can use the same optical path. The reader and tweezers canbe scanned together so that they are always in the same positionrelative to each other. For example, if the tweezers are turned on atspot-1, any bead contacted by the tweezers will be pulled into thecenter of the trap, ensuring an accurate quantitative measure of theassay label intensity. After reading the first bead, the tweezers areturned off to release it, and the scanner advances to the right just farenough to prevent the first bead from being retrapped before thetweezers are turned on again and then moved immediately to spot-2. Inthe process, any bead contacted by the tweezers would be trapped andbrought to spot-2, where it is read. Choosing a scan distance thatcorresponds to the average interbead spacing can minimize bead loss frommultiple beads occurring between sampling points.

Alternatively, the optical tweezers can be focused within the solutionaway from the surface of the well. As the tweezers are turned on andoff, the solution is mixed, so that different beads are brought into thedetection region and held while they are scanned. In anotheralternative, the optical tweezers can be focused in only one dimension,i.e., to a line rather than a spot, thus creating a linear trap region.This type of system can be used to sweep beads into distinct lines thatcan be scanned by a “line scanning” bead reader.

In another preferred embodiment, the photo-transferable coding agentscomprise a mixture of different photocleavable nanocrystals conjugates,each with distinct spectral properties. The nanocrystals are coated witha surface such as an amine reactive polymer which allows covalentbonding of NHS-PC-biotin. The PC-biotin is used to link the nanocrystalsto streptavidin-coated beads. Different compounds are attached todifferent coded beads using photocleavable conjugates described in thisinvention. The beads are allowed to directly contact the second surface.The beads are then illuminating causing photo-release and deposition ofthe compound and coding agents in the immediate vicinity of the bead.The beads are then removed from the surface leaving spots on secondsurface containing both the transferred compound and coding agents.Since the nanocrystals used in this embodiment contain amine reactivegroups they will react with a variety of surfaces. The identity of thecompound or compound mixture deposited in each spot is then determinedby the spectral properties of the photo-transferred quantum dots.

Detection of Biomarkers

An additional embodiment of the invention is directed to the detectionof biomarkers in a heterogeneous biological mixture including but notlimited to blood, serum, stool, tissue, prenatal samples, fetal cells,nasal cells, urine, saliva and cerebrospinal fluid. Biomarkers cancomprise of a variety of biomolecules or biomolecular complexesincluding proteins, nucleic acids, carbohydrates, steroids andcombinations thereof. Biomarkers can also comprise of specific types ofcells including but not limited to pathogens, bacteria, viruses, tissuecells, blood cells, colonocytes, fetal cells and tumor cells.

In one preferred embodiment, beads contain a coupling agent whichselectively binds to the biomarker. The coupling agent is linked to thebead through a photocleavable conjugate. The beads are allowed tocontact the heterogeneous sample which can contain the biomarker,separated from the heterogeneous sample and allowed to directly contactthe second surface. The beads are then illuminated at preferredwavelengths which causes photo-transfer of the biomarker in a modifiedor unmodified form to the second surface. Conventional methods are thenused to detect the presence of the biomarkers deposited on the secondsurface. Detection methods can include but are not limited to absorptionspectroscopy, fluorescence spectroscopy, fluorescent resonance energytransfer, Raman spectroscopy, mass spectrometry, addition of a labeledantibody directed against the biomarker or addition to a fluorescentlabel which selectively interacts with the biomarker and not theaffinity agent.

In another preferred embodiment, a plurality of beads comprisingseparately different coupling agents, each of which selectively binds todifferent biomarkers. The coupling agents are linked to the bead throughone or more types of photocleavable conjugates. The beads are allowed tocontact the heterogeneous sample which can contain one or more of thebiomarkers, separated from the heterogeneous sample and allowed todirectly contact the second surface. The beads are then illuminated atpreferred wavelengths which causes photo-transfer of the biomarkers in amodified or unmodified form to the second surface. Conventional methodsare then used to detect the presence of the different biomarkersdeposited on the second surface. Detection methods can include but arenot limited to absorption spectroscopy, fluorescence spectroscopy,fluorescent resonance energy transfer, Raman spectroscopy, massspectrometry, addition of a labeled antibody directed against thebiomarker or addition to a fluorescent label which selectively interactswith the biomarker and not the affinity agent.

In another preferred embodiment, a plurality of beads comprise differentcoupling agents which selectively bind to different biomarkers. Thecoupling agents are linked to the bead through one or more types ofphotocleavable conjugates. The beads are allowed to contact theheterogeneous sample which can contain one or more of the biomarkers,separated from the heterogeneous sample and allowed to directly contactthe second surface. The beads are then illuminated at preferredwavelengths which causes photo-transfer of the biomarkers in a modifiedor unmodified form to the second surface. Conventional methods are thenused to detect the presence of the different biomarkers deposited on thesecond surface. Detection methods can include but are not limited toabsorption spectroscopy, fluorescence spectroscopy, fluorescentresonance energy transfer, Raman spectroscopy, mass spectrometry,addition of a labeled antibody directed against the biomarker oraddition to a fluorescent label which selectively interacts with thebiomarker and not the affinity agent.

In another preferred embodiment, a plurality of beads comprise differentcoupling agents which selectively bind to different biomarkers. Inaddition, the beads comprise coding agents which allow theidentification of the beads and said coupling agents. The couplingagents are linked to the bead through one or more types ofphotocleavable conjugates. The beads are allowed to contact theheterogeneous sample which can contain one or more of the biomarkers,separated from the heterogeneous sample and allowed to directly contactthe second surface. The beads are then illuminated at preferredwavelengths which causes photo-transfer of the biomarkers in a modifiedor unmodified form to the second surface. The coding agents are thenused to determine the identity of the photo-transferred biomarker.

It is not intended that the present invention be limited to anyparticular type of coupling agent or biomarkers. Examples of usefulcoupling agents include molecules such as haptens, immunogenicmolecules, biotin and biotin derivatives, and fragments and combinationsof these molecules. For example, coupling agents can enable theselective binding or attachment of newly formed nascent proteins tofacilitate their detection or isolation. Coupling agents may containantigenic sites for a specific antibody, or comprise molecules such asbiotin which is known to have strong binding to acceptor molecules suchas streptavidin.

In addition, biomarkers may be (but are not limited to) small organicmolecules, proteins, nucleic acids, carbohydrates and combinationsthereof which are distinctive or change their concentrations in responseto a disease, therapeutic or other stimulus. Examples include compoundssometimes found in an increased amount in the blood, other body fluids,or tissues and that may suggest the presence of some types of cancer.Biomarkers include CA 125 (ovarian cancer), CA 15-3 (breast cancer), CEA(ovarian, lung, breast, pancreas, and GI tract cancers), and PSA(prostate cancer) also called tumor markers.

A variety of coupling agents are available that can be used toselectively bind biomarkers. In many case, the biomarker will haveantigenic properties reflecting one or more antigenic sites which willinteract with antibodies, both polyclonal and monoclonal, directed atthe particular antigenic site or sites on the biomarker.

In one embodiment, the attachment of the coupling agent to the beadoccurs through a photocleavable conjugate. There are a variety ofcompositions which can be used to achieve such attachments. For example,photocleavable biotin may be covalently linked to a component of thecoupling agent. Photocleavable biotin contains a photoreactive moietywhich comprises a phenyl ring derivatized with functionalitiesrepresented in FIG. 12 in U.S. Pat. No. 5,922,858 specificallyincorporated here by reference by X, Y and Z where X allows linkage ofPCB to the bimolecular substrate through the reactive group X′. Examplesof X′ include Cl, O—N-hydroxysuccinimidyl, OCH.sub.2 CN, OPhF.sub.5,OPhCl.sub.5, N.sub.3. Y represents a substitution pattern of a phenylring containing one or more substitutions such as nitro or alkoxyl. Thefunctionality Z represents a group that allows linkage of thecross-linker moiety to the photoreactive moiety.

The photoreactive moiety has the property that upon illumination, itundergoes a photoreaction that results in cleavage of the PCB moleculefrom the substrate. If the coupling agent is an antibody this can occurthrough a covalent bond to one or more amino acids present in theantibody. The presence of the photocleavable biotin will allow highaffinity binding of the antibody coupling agent to avidin moleculescoated onto a bead. In addition to beads, such suitable surfaces includeresins for chromatographic separation, plastics such as tissue culturesurfaces for binding plates, microtiter dishes, ceramics and glasses,particles including magnetic particles, polymers, quantum dots,nanocrystals and other matrices.

One example of an antigenic site which illustrates the methods of thisinvention and can be present on a specially prepared biomarker isdansyllysine (FIG. 5 of U.S. Pat. No. 6,596,481 specificallyincorporated here by reference). Antibodies which interact with thedansyl ring are commercially available (Sigma Chemical; St. Louis, Mo.)or can be prepared using known protocols such as described inAntibodies: A Laboratory Manual (E. Harlow and D. Lane, editors, ColdSpring Harbor Laboratory Press, 1988) which is hereby specificallyincorporated by reference. Many conventional techniques exist whichwould enable proteins containing the dansyl moiety to be separated fromother proteins on the basis of a specific antibody-dansyl interaction.For example, the antibody could be immobilized onto the packing materialof a chromatographic column. This method, known as affinity columnchromatography, accomplishes protein separation by causing the targetprotein to be retained on the column due to its interaction with theimmobilized antibody, while other proteins pass through the column. Thetarget protein is then released by disrupting the antibody-antigeninteraction. Specific chromatographic column materials such asion-exchange or affinity Sepharose, Sephacryl, Sephadex and otherchromatography resins are commercially available (Sigma Chemical; St.Louis, Mo.; Pharmacia Biotech; Piscataway, N.J.).

Separation can also be performed through an antibody-dansyl interactionusing other biochemical separation methods such as immunoprecipitationand immobilization of the antibodies on filters or other surfaces suchas beads, plates or resins. For example, protein could be isolated bycoating magnetic beads with a protein-specific antibody. Beads areseparated from the extract using magnetic fields. A specific advantageof using dansyllysine as an affinity marker is that once a protein isseparated it can also be conveniently detected because of itsfluorescent properties.

In addition to antibodies, a variety of other coupling agents areenvisioned which can be coupled to beads through a photocleavableconjugate. One example are aptamers, which comprise single-strandednucleic acids that form three-dimensional structures which speciallybind to target molecules with high affinity and specificity (Mayer G,Grättinger M, and Blind M. Aptamers: Multifunctional tools for targetvalidation and drug discovery. DrugPlus international, 2003, Nov.-Dec.,6-10). A wide range of applications which normally use monoclonalantibodies can be substituted with aptamers. However, unlike antibodieswhich are proteins and will interact and stain with similar propertiesto biomarkers comprising (completely or in part) polypeptides, aptamerswill not, thereby allowing detection of the bound biomarker. Incontrast, detection of a biomarker using antibodies normally requires asecond antibody. While this sandwich approach to detection of antigensis widely used in a variety of applications, the requirement of twoantibodies which interact with the antigen is often difficult to achievewhile maintaining strong binding and selectivity.

In one preferred embodiment, an aptamer which is selective for aspecific biomarker is linker using a photocleavable conjugate to a bead.The bead is then allowed to contact a heterogeneous sample which couldpotentially contain the biomarker. The bead is then isolated and allowedto directly contact the surface. The beads are then illuminated causingphoto-transfer of the biomarker-aptamer complex from each bead to thesurface. The beads are then removed from the surface. The presence orabsence of a biomarker is then determined using a dye which labels thebiomarker selectively.

Mass Spectrometry

Another preferred embodiment of this invention is directed at analysisof target molecules by mass spectrometry. Mass spectrometry (MS) hasbecome increasingly attractive as an analytical technique in biomedicalresearch. Matrix assisted laser desorption time of flight massspectrometry (MALDI-TOF MS) is now the core technology underlying theproteomics field because this method can quickly and accurately measurethe masses of peptides in a mixture. Mass spectrometry also holdssubstantial potential for the rapid screening of disease causing geneticdefects and the discovery of biomarkers (Koster, H., Tang, K., Fu, D.J., Braun, A., van den Boom, D., Smith, C. L., Cotter, R. J., andCantor, C. R. (1996) Nat Biotechnol 14, 1123-1128). Importantly, veryhigh throughputs are obtained because separation times are measured inmicroseconds rather than minutes or hours for conventional methods suchas gel electrophoresis (Ross, P., Hall, L., Smimov, I., and Haff, L.(1998) Nat Biotechnol 16, 1347-1351).

Mass spectrometry can be of great value in the detection and discoveryof biomarkers, provided methods can be developed that can be used torapidly isolate biomarkers from heterogeneous mixtures in a formsuitable for mass spectrometric analysis. Methods which can isolatemultiple biomarkers from a biological sample in a form suitable for massspectrometric analysis are particularly advantageous due to the rapidability of mass spectrometry to analyze each sample.

A variety of methods exist for selective absorption of biomolecules on aMALDI substrate from a heterogeneous mixture. Many of these methodsdepend on selective binding of molecules with particular physicalproperties such as hydrophobicity or hydrophilic to the surface. Othermethods involve selective binding through coupling agents present on thesurface of the MALDI substrate or on beads. Additional methods utilizeaffinity chromatography to select particular molecules from aheterogeneous mixture. However, these methods all suffer from variousdegrees of non-specific binding of non-target biomolecules to theaffinity medium and ultimately deposition on the MALDI substrate. Thisproblem can be particularly complicated when fingerprint analysis of thebiomarker is performed via proteolysis such as tryptic digestion wellknown in the field of mass spectrometry. In this case, a protein isproteolyzed into smaller fragments and the molecular mass of theproteolytic fragments used to identify the target protein or targetcomplex.

These problems are significantly reduced through the use of the methodsand compositions of the present invention. Because only molecules thatare linked to a surface (B) through a photocleavable conjugate arereleased to bind to a second MALDI surface (A), non-specific absorptionis greatly reduced. Furthermore the use of a plurality of beadscontaining different coupling agents (e.g. antibodies) provides a meansto perform multiplex biomarker detection. Alternatively, beads withcommon coupling agents can also be advantageously utilized in manyapplications as described later.

In one preferred embodiment, beads contain a coupling agent whichselectively binds to a biomarker which may be present in a heterogeneousmixture. The said coupling agent is linked to the bead through aphotocleavable conjugate. The beads are allowed to contact theheterogeneous sample which can contain the biomarker, separated from theheterogeneous sample and allowed to directly contact the MALDI secondsurface. The beads are then illuminated at preferred wavelengths whichcause photo-transfer of the biomarker in a modified or unmodified formto the MALDI substrate. Mass spectrometry is then used to detect thepresence of the biomarkers deposited on the second surface.

In another preferred embodiment, a plurality of beads contain differentcoupling agents which selectively bind to different biomarkers. Inaddition, the beads contain coding agents which allow the identificationof the beads and said coupling agents. The said coupling agents arelinked to the bead through one or more types of photocleavableconjugates. The beads are allowed to contact the heterogeneous samplewhich can contain one or more of the biomarkers, separated from theheterogeneous sample and allowed to directly contact the second surface.The beads are then illuminated at preferred wavelengths which causesphoto-transfer of the biomarkers in a modified or unmodified form to thesecond surface. The coding agents are then used to determine theidentity of the photo-transferred biomarker.

In one embodiment the method used to identify the photo-transferredcoding agents, biomarker or biomarker complex is based on the use ofmass spectrometry. For example, small polypeptides can serve as codingagents if they have unique masses compared to other coding agents. Themass of the proteolytic fragments from a transferred substance such as abiomarker or biomarker complex can also be used in order to uniquelyidentify it.

It is to be understood that the present invention is not limited to aparticular MALDI substrate. However, some MALDI substrates are preferredbecause of the ability to adhere to a variety of biomarkers. In onepreferred embodiment, a MALDI substrate is utilized which containschemically reactive groups which form covalent bonds with a variety ofbiomolecules. One method which could be used to activate MALDI platescoated with gold, consists of soaking the surface with 4 mM solution of(Dithiobis-succinimidyl-proprionate (DTSP) in DMSO which results in theabsorption of the N-succinimidyl-3-thiopropionate Darder, M., Takada,K., Pariente, F., Lorenzo, E., and Abruna, H. D. (1999) Anal Chem 71,5530-5537. These groups will result in a MALDI plate surface which isexpected to be highly reactive with amide groups in proteins. Anotherapproach is to coat the MALDI plate with a nitrocellose surface. Such asurface is well known as advantageous for protein absorption. In onereport (Miliotis, T., Marko-Varga, G., Nilsson, J., and Laurell, T.(2001) J Neurosci Methods 109, 41-46), nitrocellulose was coated on aMALDI target plate. An acetone solution consisting of matrix (10 mg/ml)and nitrocellose membrane (0.5 mg/ml) was precoated as thin film on thetargets using an air-brush device. (Miliotis, T., Kjellstrom, S.,Nilsson, J., Laurell, T., Edholm, L. E., and Marko-Varga, G. (2002)Rapid Commun Mass Spectrom 16, 117-126.).

Photo-Release of Targets from Beads for Improved Detection

An additional embodiment of the invention is directed to the detectionof target molecules by a biomolecular detection device such as amicroarray-based device. Target molecules are normally present inheterogeneous biological mixture including but not limited to blood,serum, stool, tissue, prenatal samples, fetal cells, nasal cells, urine,saliva and cerebrospinal fluid. Targets can also comprise agents in theenvironment including but not limited to allergens, toxins, pathogens,biowarfare agents. Environmental targets can be present in air, liquid,soil, surfaces, solids that are part of environment. Targets cancomprise a variety of biomolecules or biomolecular complexes includingbiomarkers, proteins, nucleic acids, carbohydrates, steroids andcombinations thereof. Targets can also consist of specific types ofcells including but not limited to pathogens, bacteria, viruses, tissuecells, blood cells, colonocytes, fetal cells and tumor cells. Typically,targets are detected by their interaction with probes which are used aspart of the target detection process. For example, probes are depositedon microarray substrates for subsequent possible interaction withtargets in the sample comprising a heterogeneous mixture.

A major limitation of current microarray technology and more generallybiomolecular detection is the difficulty of detecting with sufficientsensitivity and accuracy low levels of target molecules, especially whenpresent in heterogeneous mixtures. For example, in the field of medicaldiagnostics the target biomolecule, which often serves as a biomarkerinclude but are not limited to proteins, antigens, antibodies, cells andnucleic acid. These molecules are often present at very lowconcentrations in the presence of a complex mixture of otherbiomolecules.

In the case of basic research, a similar need exists for increasedsensitivity to detect target biomolecules that are present in aheterogeneous mixture. For example, it is often essential to monitor thechange in the level of biological molecules in specific cells, cellcultures or tissues in response to various stimuli. Furthermore, thevolume of the fluid analyzed by the biomolecular detection device suchas a microarray is often small, in the range of 10-100 microliters, thuslimiting the number of target molecules available for binding to theprobes. In the case of portable diagnostic devices such as glucosemeters even smaller volumes, e.g. 1 microliters of blood are analyzed.The low volume and low concentration of target molecules can necessitatethe use of time consuming, expensive techniques in order to concentratethe target molecules without destroying their activity. These methodsare normally not compatible with the need for rapid measurements oftargets.

In one preferred embodiment of this invention, the targets present in aheterogeneous mixture are bound to the bead using a photocleavableconjugate. The beads are then isolated and concentrated in a preferredsolution. The target is then photo-released from the bead in a modifiedor unmodified form and the beads removed. The photo-released targetmolecules are then allowed to interact with the probes.

In another preferred embodiment of the invention, the targets present ina heterogeneous mixture are bound to the bead using a photocleavableconjugate. The beads are then isolated and concentrated in a preferredsolution. The target is then allowed to interact with the probes andsubsequently photo-released from the bead in a modified or unmodifiedform.

In another preferred embodiment of the invention, the targets present ina heterogeneous mixture are bound to the bead using a photocleavableconjugate. The beads are then isolated and concentrated in a preferredsolution which is introduced to the biomolecular detection device.

It is to be understood that in these embodiments, the method is notlimited by the nature of the target. Targets can consist but not limitedto compounds, molecules, biomolecules, macromolecules and cells whichare ordinarily present in a heterogeneous mixture such as blood seraand. Molecules and macromolecules comprise but are not limited toproteins, peptides, amino acids, amino acid analogs, nucleic acids,nucleosides, nucleotides, lipids, vesicles, detergent micelles, cells,virus particles, fatty acids, saccharides, polysaccharides, inorganicmolecules and metals.

The invention is also not limited by the nature of the beads, whichcould be composed of a variety of materials including but not limited toorganic or inorganic molecules, polymer, solid-state materials such asmetals or semiconductors, biological materials, sol gels, colloids,glass, paramagnetic and magnetic materials, electrostatic materials,electrically conducting materials, insulators, fluorescent materials,absorbing material and combinations thereof. The beads may also vary insize, shape and density. For example beads may range in size from 20nanometers to hundreds of microns depending on the application and spotsize desired for different applications. The beads may also bepolydisperse in regards to size, shape, material composition, optical,magnetic, electrical properties. Beads may also consist of aggregates ofsmaller beads.

In one preferred embodiment the target is a specific IgE antibody whichis present in blood. The beads contain an anti-IgE antibody attached tothe bead through a photocleavable conjugate. The beads are allowed tocontact the blood sample and then are isolated and concentrated in abuffer solution. The IgE-anti IgE complex is then photo-released fromthe beads into the buffer solution. The solution is then introduced intothe microarray chamber for subsequent detection of IgE molecules whichhave a specificity to interact with specific probe allergens on thearray surface.

In another preferred embodiment the target is a specific IgE antibodywhich is present in blood. The beads contain a specific allergen whichserves as an antigen for the specific IgE target molecules. The allergenis attached to the bead through a photocleavable conjugate. The beadsare allowed to contact the blood sample and then are isolated andconcentrated in a buffer solution. The IgE-allergen complex is thenphoto-released from the beads into the buffer solution. The solution isthen introduced into the microarray device for subsequent detection ofthe IgE allergen complex by a probe molecule. Probe molecules canconsist of an antibody directed against the allergen.

In another preferred embodiment the target is a protein biomarkerpresent in blood. The beads contain an antibody directed against thebiomarker which is attached to the bead through a photocleavableconjugate. The beads are allowed to contact the blood sample and thenare isolated and concentrated in a buffer. The biomarker-antibodycomplex is then photo-released from the bead into the buffer solutionand the beads removed. The solution is then introduced into themicroarray device for subsequent detection of biomarker-antibodiescomplex by specific probe antibodies present on the microarray surface.

It is to be understood that the invention is not limited by the numbertargets detected. For example, a plurality of beads can be prepared suchthat some beads are coated with antibodies directed towards target X,while other beads contain antibodies directed towards target Y. In thegeneral case where the microarray is designed to detect N differenttargets, N different types of beads, each bead type with a correspondingantibody, are prepared. Each of the antibodies are attached to the beadsthrough a photocleavable conjugate using for example photocleavablebiotin. In addition to antibodies, aptamers can be utilized for captureof the target molecule.

Photocleavable Conjugates

Probes as referred to herein, as those compounds being deposited on asurface using the agents, conjugates and methods of the invention.Targets are referred to herein as those compounds detected using theagents, conjugates and methods of the invention. Substrates, as referredto herein, are those compounds which are covalently attached to thebioreactive agent. Substrates may also be referred to as targets whenthe target being identified specifically binds to the bioreactive agent.

Photocleavable conjugates are described in U.S. Pat. No. 5,986,076,which is specifically incorporated by reference, and variations thereofdescribed in U.S. Pat. Nos. 6,057,096 and 6,589,736 which are alsospecifically incorporated by reference. Photocleavable conjugatescomprise bioreactive agents photocleavable coupled to substrates.Conjugates have the property that they can be selectively cleaved withelectromagnetic radiation to release the substrate. Substrates are thosechemicals, compounds, macromolecules, cells and other compounds whichare or can be used to couple probes or targets. Substrates that areselectively cleaved from conjugates may be modified by photocleavage ormay be released from the conjugate completely unmodified byphotocleavage. Substrates may be coupled with agents, uncoupled andrecoupled to new agents at will.

Agents of the invention comprise a detectable moiety and a photoreactivemoiety, and can be covalently coupled to a variety of substrates to forma photocleavable conjugate. A covalent bond between agent and substratecan be created from a wide variety of chemical moieties includingamines, hydroxyls, imidazoles, aldehydes, carboxylic acids, esters andthiols. Agent-substrate combinations are referred to herein asconjugates. Through the presence of the detectable moiety, conjugatescan be quickly and accurately bound to a bead or used to isolate a probeor target. Further, these conjugates are selectively cleavable whichprovides unique advantages in isolation procedures and release of theprobe or target for subsequent deposition on the array surface ordetection. Substrate can be separated from agent quickly andefficiently. Complex technical procedures and highly trained experts arenot required. New attachment and separation procedures do not need to bedeveloped for every new probe or target to be used with a microarray.Following isolation of probe or target, it is a relatively simple matterto treat the conjugate with electromagnetic radiation and release thesubstrate. Released substrate is preferably functionally active andstructurally unaltered. Nevertheless, minor chemical alterations in thestructure may occur depending on the point of attachment. It isgenerally preferred that such alterations not affect functionalactivity. However, when functional activity does not need to bepreserved, such changes are of no considerations and may even be usefulto for delivering probe or target to microarray by methods of theinvention.

It is not intended that the present invention be limited to the natureof the particular photocleavable conjugates. A variety of photocleavableconjugates are contemplated, including conjugates that photocleave overa variety of infrared, visible and UV wavelengths. Nonetheless, comparedto many other photocleavable conjugates, several have been empiricallyfound to have very efficient quantum yields for photocleavage and arenot sensitive under normal laboratory conditions to photocleavage. Theyinclude reagents and compounds described in U.S. Pat. No. 5,986,076“Photocleavable agents and conjugates for the detection and isolation ofbiomolecules” and U.S. Pat. Nos. 6,057,096 and 6,589,736, herebyincorporated by reference.

Useful substrates are any chemical, macromolecule or cell that can beattached to a bioreactive agent. Examples of useful substrates includeproteins, peptides, amino acids, amino acid analogs, nucleic acids,nucleosides, nucleotides, lipids, vesicles, detergent micells, cells,virus particles, fatty acids, saccharides, polysaccharides, inorganicmolecules and metals. Substrates may also comprise derivatives andcombinations of these compounds such as fusion proteins,protein-carbohydrate complexes and organo-metallic compounds. Substratesmay also be pharmaceutical agents such as cytokines, immune systemmodulators, agents of the hematopoietic system, recombinant proteins,chemotherapeutic agents, radio-isotopes, antigens, anti-neoplasticagents, enzymes, PCR products, receptors, hormones, vaccines, haptens,toxins, antibiotics, nascent proteins, synthetic pharmaceuticals andderivatives and combinations thereof. Substrates may also be aptamerscomprised of nucleic acid.

Substrates may be probes or targets or part of the probes or targetssuch as an amino acid in the synthesis of nascent polypeptide chainswherein substrates may be amino acid or amino acid derivative whichbecomes incorporated into the growing peptide chain. Substrates may alsobe nucleotides or nucleotide derivatives as precursors in the synthesisof a nucleic acid. Constructs useful in creating syntheticoligonucleotide conjugates may contain phosphoramidites or derivativesof DATP, dCTP, dTTP and dGTP, and also ATP, CTP, UTP and GTP. Resultingnucleic acid-conjugates can be used in hybridization technology as bothtargets and probes.

Photocleavage of conjugates of the invention should preferably notdamage released substrate or impair substrate activity. Proteins,nucleic acids and other protective groups used in peptide and nucleicacid chemistry are known to be stable to most wavelengths of radiationabove 300 nm. PCB carbamates, for example, undergo photolysis uponillumination with long-wave UV light (320-400 nm), resulting in releaseof the unaltered substrate and carbon dioxide. The yield and exposuretime necessary for release of substrate photo-release are stronglydependent on the structure of photoreactive moiety. In the case ofun-substituted 2-nitrobenzyl PCB derivatives the yield of photolysis andrecovery of the substrate are significantly decreased by the formationof side products which act as internal light filters and are capable ofreacting with amino groups of the substrate. In this case, illuminationtimes vary from about 1 minute to about 24 hours, preferably less than 4hours, more preferably less than two hours, and even more preferablyless than one hour, and yields are between about 1% to about 95% (V. N.R. Pillai, Synthesis 1, 1980). In the case of alpha-substituted2-nitrobenzyl derivatives (methyl, phenyl), there is a considerableincrease in rate of photo-removal as well as yield of the releasedsubstrate (J. E. Baldwin et al., Tetrahedron 46:6879, 1990; J. Nargeotet al., Proc. Natl. Acad. Sci. USA 80:2395, 1983).

It is not intended that the present invention be limited to the natureof the attachment of the photocleavable conjugate to a bead surface.Examples of the chemical structure of conjugates of the inventioninclude: a structure described in U.S. Pat. No. 5,986,076 (Structure 5)specifically incorporated here by reference wherein SUB comprises asubstrate; R.sub. 1 and R.sub.2 are selected from the group consistingof hydrogen, alkyls, substituted alkyls, aryls, substituted aryls,—CF.sub.3, —NO.sub.2, —COOH and —COOR, and may be the same or different;A is a divalent functional group selected from the group consisting of—O—, —S— and —NR.sub.1; Y comprises one or more polyatomic groups whichmay be the same or different; V comprises one or more optionalmonoatomic groups which may be the same or different; Q comprises anoptional spacer moiety; m1 and m2 are integers between 1-5 which can bethe same or different; and D comprises a selectively detectable moietywhich is distinct from R.sub.1 and R.sub.2.

As discussed above, the polyatomic group may be one or more nitrogroups, alkyl groups, alkoxyl groups, or derivatives or combinationsthereof. The optional monoatomic group may be one or more fluoro,chloro, bromo or iodo groups, or hydrogen. The polyatomic and monoatomicgroups and the chemical moieties at R.sub. 1 and R.sub.2 may effect thephotocleavage reaction such as the frequency of radiation that willinitiate photocleavage or the exposure time needed to execute a cleavageevent. The spacer moiety (Q) may be a branched or unbranched hydrocarbonor a polymeric carbohydrate and is preferably represented by the formuladescribed in U.S. Pat. No. 5,986,076 (Structure 6 specificallyincorporated here by reference) wherein W and W′ are each selected fromthe group consisting of —CO—, —CO—NH—, —HN—CO—, —NH—, —O—, —S— and—CH.sub.2-, and may be the same or different; and n1 and n2 are integersfrom 0-10 which can be the same or different and if either n1 or n2 iszero, then W and W′ are optional. Specific examples of conjugates of theinvention are depicted in FIG. 8 of U.S. Pat. No. 5,986,076 specificallyincorporated here by reference.

In addition, it is not intended that the invention be limited to onlybead surfaces. Conjugates of the invention may be attached to a solidsupport via the detectable moiety, the substrate or any other chemicalgroup of the structure. The solid support may comprise constructs ofglass, ceramic, plastic, metal or a combination of these compounds. Inaddition to beads and microbeads, useful structures and constructsinclude plastic structures such as microtiter plate wells or the surfaceof sticks, paddles, alloy and inorganic surfaces such as semiconductors,two and three dimensional hybridization and binding chips, and magneticbeads, chromatography matrix materials and combinations of thesematerials.

Nascent Proteins

One of the preferred embodiments of the invention relates to thedeposition of protein on a surface. In one application of thisembodiment, photocleavable biotin (PCB) is reacted with a proteinthrough the formation of covalent bonds with specific chemicals groupsof the protein thereby forming a conjugate. The protein may be eitherthe target to be isolated or detected or a probe for the target proteinsuch as an antibody. The target protein can then be isolated usingstreptavidin affinity methodology. For example beads that are coatedwith streptavidin are used to capture the target or probe protein. Thisprotein is then photo-released for subsequent transfer to a surface suchas part of a microarray device.

Another application of this embodiment is directed to the use ofphotocleavable biotin to deposit nascent proteins that can be createdfrom in vitro or in vivo protein synthesis on a surface. Basically, inthis embodiment, photocleavable biotins are synthesized and linked toamino acids (PCB-amino acids) containing special blocking groups. Theseconjugates are charged to tRNA molecules and incorporated into peptidesand proteins using a translation or coupled transcription/translationsystem. PCB-amino acids of the invention have the property that onceilluminated with light, a photocleavage occurs that produces a nativeamino acid plus the free biotin derivative. Such proteins can bephoto-released in a structurally and/or functionally unaltered form forcontact photo-transfer to a surface or for detection by a biomoleculardevice.

The detailed procedure for the production of photocleavable biotin aminoacids and their incorporation into the nascent proteins involves a fewbasic steps. First, photocleavable biotin is synthesized and linked toan amino acid with an appropriate blocking group. These PCB-amino acidconjugates are charged to tRNA molecules and subsequently incorporatedinto nascent proteins in an in vivo or in vitro translation system.Alternatively, a tRNA molecule is first charged enzymatically with anamino acid such as lysine which is then coupled to a reactive PCB.Nascent proteins are separated and isolated from the other components ofsynthesis using immobilized streptavidin. Photocleavage ofPCB-streptavidin complex from the nascent protein generates a pure andnative, nascent protein.

PCB is attached to an amino acid using, for example, the side-chaingroups such as an amino group (lysine), aliphatic and phenolic hydroxylgroups (serine, threonine and tyrosine), sulfydryl group (cysteines) andcarboxylate group (aspartic and glutamic acids) (FIG. 9 of U.S. Pat. No.5,986,076 specifically incorporated here by reference). Synthesis can beachieved by direct condensations with appropriately protected parentamino acids. For example, lysine side chain amino group can be modifiedwith PCB by modification of the epsilon amino group. The synthesis of,for example, PCB-methionine involves primarily alpha amino groupmodification. PCB-methionine can be charged to an initiator tRNA whichcan participate in protein synthesis only at initiation sites whichresults in single PCB incorporation per copy of the nascent protein.

One method for incorporation of a photocleavable biotin amino acid intoa nascent protein involves misaminoacylation of tRNA. Normally, aspecies of tRNA is charged by a single, cognate native amino acid. Thisselective charging, termed here enzymatic aminoacylation, isaccomplished by enzymes called aminoacyl-tRNA synthetases and requiresthat the amino acid to be charged to a tRNA molecule be structurallysimilar to a native amino acid. Chemical misaminoacylation can be usedto charge a tRNA with a non-native amino acids such as photocleavableamino acids. The specific steps in chemical misaminoacylation of tRNAsare depicted in FIG. 10 of U.S. Pat. No. 5,986,076 specificallyincorporated here by reference.

As shown, tRNA molecules are first truncated to remove the 3′-terminalresidues by successive treatments with periodate, lysine (pH 8.0) andalkaline phosphate (Neu et al., J. Biol. Chem. 239:2927-34, 1964).Alternatively, truncation can be performed by genetic manipulation,whereby a truncated gene coding for the tRNA molecule is constructed andtranscribed to produce truncated tRNA molecules (Sampson et al., Proc.Natl. Acad. Sci. USA 85:1033, 1988). Second, protected acylateddinucleotides, pdCpA, are synthesized (Hudson, J. Org. Chem. 53:617,1988; E. Happ, J. Org. Chem. 52:5387, 1987). PCB-amino acids blockedappropriately at their side chains and/or at a-amino groups, usingstandard protecting groups like Fmoc, are prepared and coupled with thesynthetic dinucleotide in the presence of carboxy group activatingreagents. Subsequent deprotection of Fmoc groups yields aminoacylateddinucleotide.

Third, the photocleavable biotin amino acid is ligated to the truncatedtRNA through the deprotected dinucleotide. The bond formed by thisprocess is different from that resulting from tRNA activation by anaminoacyl-tRNA synthetase, however, the ultimate product is the same. T4RNA ligase does not recognize the O-acyl substituent, and is thusinsensitive to the nature of the attached amino acid (FIG. 10 of U.S.Pat. No. 5,986,076 specifically incorporated here by reference).Misaminoacylation of a variety of non-native amino acids can be easilyperformed. The process is highly sensitive and specific for thestructures of the tRNA and the amino acid.

Aminoacylated tRNA linked to a photocleavable biotin amino acid can alsobe created by employing a conventional aminoacyl synthetase toaminoacylate a tRNA with a native amino acid or by employing specializedchemical reactions which specifically modify the native amino acidlinked to the tRNA to produce a photocleavable biotin aminoacyl-tRNAderivative. These reactions are referred to as post-aminoacylationmodifications. Such post-aminoacylation modifications do not fall underthe method of misaminoacylation, since the tRNA is first aminoacylatedwith its cognate described amino acid.

In contrast to chemical aminoacylation, the use of post-aminoacylationmodifications to incorporate photocleavable biotin non-native aminoacids into nascent proteins is very useful since it avoids many of thesteps including in misaminoacylation. Furthermore, many of thephotocleavable biotin derivatives can be prepared which have reactivegroups reacting specifically with desired side chain of amino acids. Forexample, postaminoacylation modification of lysine-tRNA.sup.Lys, anN-hydroxysuccinimide derivative of PCB can prepared that would reactwith easily accessible primary epsilon amino and minimize reactionsoccurring with other nucleophilic groups on the tRNA or alpha-aminogroups of the amino acylated native amino acid. These other non-specificmodifications can alter the structure of the tRNA structure and severelycompromise its participation in protein synthesis. Incomplete chainformation could also occur when the alpha-amino group of the amino acidis modified. Post-aminoacylation modifications to incorporatelysine-biotin non-native amino acids into nascent proteins has beendemonstrated (Promega's Transcend tRNA; Promega; Madison, Wis.) used forthe detection of nascent protein containing biotin using Western Blotsfollowed by enzymatic assays for biotin (T. V. Kurzchalia et al., Eur.J. Biochem. 172:663-68, 1988). However, these biotin derivatives are notphotocleavable which, in the case of NHS-derivatives of PCB, allows thebiotin linkage to the lysine to be photochemically cleaved.

PCB-amino acids can also be incorporated into polypeptide by means ofsolid-support peptide synthesis. First, PCB-amino acids are derivatizedusing base labile fluorenylmethyloxy carbonyl (Fmoc) group for theprotection of alpha-amino function and acid labile t-butyl derivativesfor protection of reactive side chains. Synthesis is carried out on apolyamide-type resin. Amino acids are activated for coupling assymmetrical anhydrides or pentafluorophenyl esters (E. Atherton et al.,Solid Phase Peptide Synthesis, IRL Press, Oxford, 1989). Second, aminoacids and PCB are coupled and the PCB-amino acid integrated into thepolypeptide chain. Side chain PCB-derivatives, like epsilon-amino-Lys,side chain PCB-amino acid esters of Glu and Asp, esters of Ser, Thr andTyr, are used for incorporation at any site of the polypeptide.PCB-amino acids may also be incorporated in a site-specific manner intothe chain at either predetermined positions or at the N-terminus of thechain using, for example, PCB-derivatized methionine attached to theinitiator tRNA.

A wide range of polypeptides can be formed from PCB-amino acidscytokines and recombinant proteins both eukaryotic and prokaryotic (e.g.alpha-, beta- or gamma-interferons; interleukin-1, -2, -3, etc.;epidermal, fibroblastic, stem cell and other types of growth factors),and hormones such as the adrenocorticotropic hormones (ACTHs), insulin,the parathyroid hormone (bPTH), the transforming growth factor .beta.(TGF-.beta.) and the gonadotropin releasing hormone (GnRH) (M. Wilcheket al., Methods Enzymol. 184:243, 1990; F. M. Finn et al., MethodsEnzymol. 184:244, 1990; W. Newman et al., Methods Enzymol. 184:275,1990; E. Hazum, Methods Enzymol. 184:285, 1990). These hormones retaintheir binding specificity for the hormone receptor. One example is theGnRH hormone where a biotin was attached to the epsilon amino groupLys-6 through reaction of a d-biotin p-nitophenyl ester. Thisbiotinylated hormone can be used for isolation of the GnRH receptorusing avidin coated columns.

After incorporation or attachment of PCB into a protein, protein-complexor other amino acid-containing target, the target is isolated using asimple four step procedure (FIG. 11 of U.S. Pat. No. 5,986,076specifically incorporated here by reference). First, a bioreactive agent(PCB) is synthesized. Second, a substrate is coupled to the bioreactiveagent forming a conjugate. Third, target is separated from othermaterials in the mixture through the selective interaction of thephotocleavable biotin with avidin, streptavidin or their derivatives.Captured targets may be immobilized on a solid support such as magneticbeads, affinity column packing materials or filters which facilitatesremoval of contaminants. Finally, the photocleavable biotin is detachedfrom the target by illumination of a wavelength which causes thephotocleavable biotin covalent linkage to be broken. Targets aredissolved or suspended in solution at a desired concentration. In thosesituations wherein conjugate coupled targets are not attached to solidsupports, release of targets can be followed by another magnetic captureto remove magnetic particles now containing avidin/streptavidin boundbiotin moiety released form the photocleavage of PCB. Thus, a completelyunaltered protein is released in any solution chosen, in a purified formand at nearly any concentration desired.

In one embodiment of this invention, nascent proteins are produced in anin vitro or in vivo translation system using misaminoacylated tRNAs toincorporate photocleavable biotin. The nascent protein is captured usingbeads coated with streptavidin. The beads are used to contact a surfaceand illuminated with light to transfer said nascent proteins to surface.

Another embodiment of this invention is directed at constructing aproteomic microarray which is used to probe protein-proteininteractions. The conventional method of protein expressionprofiling/identification in normal and diseased states relies on the useof 2D gel electrophoresis and mass spectrometry. While this approach hasbeen invaluable in proteomics, recent studies show that approximately40% of cellular proteins, many involved in key process such astranscription control, are missed because of their low concentrations(e.g. low copy number) in the cell. In addition, 2D gel electrophoresisis a relatively slow process and not compatible with the high throughputneeded to map the vast number of protein-protein interactions that occurin the cell. It is also not suitable for use in clinical studies wherelarge numbers of patients are involved. Finally, 2D gel technology isunable to probe the function of each of the proteins comprising theproteome, a critical requirement for future progress.

As an alternative to gel electrophoresis, many researchers andcommercial companies have begun exploring the use of protein microarrays(Zhu, H., Bilgin, M., Bangham, R., Hall, D., Casamayor, A., Bertone, P.,Lan, N., Jansen, R., Bidlingmaier, S., Houfek, T., Mitchell, T., Miller,P., Dean, R. A., Gerstein, M., and Snyder, M. (2001) Science 293,2101-2105; Zhu, H., and Snyder, M. (2001) Curr Opin Chem Biol 5, 40-45).Such arrays have the advantage that they in principle can provide thehigh sensitivity and speed not available using gel electrophoresis. Inaddition, protein arrays can be used to study protein function. However,unlike DNA arrays, where oligonucleotide probes for each gene can bereadily synthesized, creating a set of capture elements for mostproteins in the proteome is significantly more difficult.

An attractive alternative is to array the proteome of a specificorganism on a chip. Such a proteome array would be highly suitable formapping protein-protein interactions, probing the function of specificproteins in the array, and discovering biomarkers of specific diseases.For example, many protein components of a particular cell are likely tointeract and in some cases enzymatically modify proteins from the array.Such interactions/modifications provide a specific profile which islikely to change between normal and diseased state. Clinical samplessuch as blood and urine are also likely to contain protein biomarkers,especially in the case of infectious disease where components of thepathogen or antibodies which are developed against it are present.

In one embodiment aimed a fabricating a proteomic array, a plurality ofnascent proteins are expressed by in vitro or in vivo protein synthesissystems and attached to beads through photocleavable conjugates. Beadsmay contain a single species of protein or a mixture of differentspecies. Beads are deposited on a surfaces using the direct contactphoto-transfer method described in this invention. The deposited nascentproteins are then used as probes for a microarray that can detectpossible interaction of specific biomolecules with the nascent proteinarray.

A major advantage of this approach is that custom made microarrays canbe rapidly produced by selecting only those beads which carry the probeswhich are desired to be used for the microarray experiment. Until thisdecision is made, proteins are stored on beads and then deposited on amicroarray substrate by direct contact photo-transfer.

In one example, an assortment of beads carrying different nascentproteins are mixed together in a small volume of buffer and allowed tocontact the microarray surface. The beads are then irradiated andremoved. This results in a set of spots on the microarray surface, eachcontaining a different protein.

Cell-Free Synthesis of Nascent Proteins on Beads for Probes, Targets andCoding Agents

One preferred embodiment of this invention is directed at the synthesisof nascent proteins on the surface of a bead, the attachment of saidprotein to said bead through a photocleavable linker and the subsequentphoto-release of said nascent protein. In this embodiment, the codingDNA or RNA (nucleic acid template) for the nascent protein is attacheddirectly to the bead along with a coupling agent for said nascentprotein. A photocleavable linker is incorporated directly into thenascent protein during its synthesis using tRNA based methods describedin this invention. Alternatively, the said coupling agent is attached tobead through a photocleavable linker. A preferred embodiment is theincorporation of the photocleavable linkers NHS-PC-biotin into saidnascent protein, whereas the coupling agent on the bead is streptavidin.A second preferred embodiment is the use of an antibody which isdirected at an epitope incorporated into said nascent proteins andencoded by the attached nucleic acid template through a photocleavablelinker such as NHS-PC-biotin which binds to streptavidin on the beadsurface. The synthesis of nascent proteins using nucleic acid templateattached to a planar surface and its subsequent capture by tetherednon-photocleavable antibodies has already been described (Ramachandran,N., Hainsworth, E., Bhullar, B., Eisenstein, S., Rosen, B., Lau, A. Y.,Walter, J. C., and LaBaer, J. (2004) Science 305, 86-90).

It is to be understood that this invention allows for a plurality ofnascent proteins to be synthesized and attached to a plurality of beads.For example, different nucleic acid templates can be attached todifferent beads thereby allowing the nascent proteins which eachtemplate codes for to become photocleavable attached to thecorresponding bead. Thus bead type A which attaches nucleic acidtemplate A and codes for nascent protein A, will capture primarilynascent protein A. Whereas, bead type B which attaches nucleic acidtemplate B and codes for nascent protein B, will capture primarilynascent protein B. Using this method, a large number of differentnascent proteins can be synthesized in a cell free mixture and becomeattached to a large number of specific beads containing thecorresponding nucleic acid template coding for said nascent protein.

In one preferred embodiment, each bead has a DNA template andstreptavidin or NeutrAvidin attached to the surface. The beads areplaced in a coupled rabbit reticulocyte transcription/translation systemsuch as sold by Promega Corp. along with tRNA which incorporatesPC-biotin into various amino acid positions in the protein as describedin U.S. Pat. No. 6,210,941 and is hereby incorporated by reference.After incubation of the beads for less than 1 hour (e.g. between 15 and45 minutes, more preferably for 30 minutes), the beads are removed fromthe rabbit reticulocyte system and washed. The beads are then depositedon Epoxy coated glass in a solution and allowed to settle on said slide.The slide is then illuminated with light with wavelengths longer than300 nm (e.g. between 300 nm and 400 nm, more preferably between 300 nmand 360 mm) for a period of time (less than one hour, more preferablyless than 30 minutes, still more preferably between 1 and 10 minutes).The beads are then washed from the slide with a stream of water.

In one preferred embodiment, the DNA template is attached to thestreptavidin through a photocleavable biotin. There are a variety ofmethods which will be known to those skilled in the area of nucleic acidchemistry to attach photocleavable biotin to DNA. One such methodinvolves incorporating photocleavable biotin at the 5′ end of the DNAwhich is chemically synthesized using a PC-biotin phosphoramidite asdescribed previously [Olejnik, J., Krzymanska-Olejnik, E., andRothschild, K. J. (1996) Nucleic Acids Research 24, 361-366] and soldcommercially by Glen Research Corporation and also described in U.S.Pat. No. 5,986,076 and hereby incorporated by reference. An alternativemethod is to utilize primers containing the 5′ PC-biotin in order toamplify a DNA sequence of interest. In both cases, after incubation ofthe beads for less than 1 hour (e.g. between 15 and 45 minutes, morepreferably for 30 minutes), the beads are removed from the rabbitreticulocyte system and washed. The beads are then deposited on a slidein a solution and allowed to settle on said slide. The slide is thenilluminated with light with wavelengths longer than 300 nm (e.g. between300 nm and 400 nm, more preferably between 300 nm and 360 nm) for aperiod of time (less than one hour, more preferably less than 30minutes, still more preferably between 1 and 10 minutes) therebyallowing both the said nascent protein and DNA template to betransferred to said slide. The beads are then removed from the slide.

The transfer of the template DNA and the nascent protein for which itcodes to the same spot on a slide provides an effective coding agent forthe nascent protein. For example, the identity of the template DNA canbe subsequently determined using a number of methods well known in thefield including the use of polymerase chain reaction, hybridizationprobes or a combination of both. One such method is part of theIllumina's SNP decoding technology and recently described [Gunderson, K.L., Kruglyak, S., Graige, M. S., Garcia, F., Kermani, B. G., Zhao, C.,Che, D., Dickinson, T., Wickham, E., Bierle, J., Doucet, D., Milewski,M., Yang, R., Siegmund, C., Haas, J., Zhou, L., Oliphant, A., Fan, J.B., Barnard, S., and Chee, M. S. (2004) Genome Res 14, 870-877].

In another preferred embodiment the nascent proteins synthesized on abead using the attached template nucleic acid are coded for using avariety of other coding agents which are attached to the bead anddescribed previously in this invention. In one embodiment, the codingagents consist of quantum dots which are specifically attached to thebead through a photocleavable linker, thereby allowing direct contactphoto-transfer of the quantum dots to a surface along with the saidnascent proteins. It is to be understood that all of the methods andcompositions described under section II can be applied equally as wellto coding for nascent proteins synthesized on a bead using an attachednucleic acid template.

In one especially preferred embodiment, the coding agent is a nascentprotein that is synthesized on the bead surface. For example, a nucleicacid which codes for one of a variety of different green fluorescentprotein can be used to produce the green fluorescent protein that servesas a coding agent.

The concept of cell-free synthesis of a protein on the surface of a beadwhich can be photo transferred to a surface is useful in a variety ofapplications including molecular diagnostics and proteomics. Onepreferred embodiment is related to the creation of a customized proteinarrays. The creation of such protein arrays are especially attractivewhen a differential gene expression analysis reveals that particulardisease related cell types exhibit abnormal gene expression. In suchcases it is highly desirable to move beyond transcriptional activity inorder to understand the basis of the disease state. In this case, thestate of individual proteins and their interactions in a diseased cellwhich correspond to those proteins coded by abnormally expressed genescan be explored.

Libraries of In Vitro Expressed Proteins

In one preferred embodiment, primer pairs are attached to individualbeads. Said primer pairs are designed to amplify specific nucleic acidsequences in a sample such as genomic DNA or mRNA using BRIDGEamplification, a solid phase PCR amplification technology also referredto as solid phase amplification (SPA). (see Promega Catolog; Adessi, C.,G. Matton, G. Ayala, G. Turcatti, J.-J. Mermod, P. Mayer, and E.Kawashima. 2000. Solid phase amplification: characterisation of primerattachment and amplification mechanisms. Nucleic Acids Res. 28:e87 andBing, D. H., C. Boles, F. N. Rehman, M. Audeh, M. Belmarsh, B. Kelley,and C. P. Adams; 1996. Bridge amplification: a solid phase PCR systemfor the amplification and detection of allelic differences in singlecopy genes In Genetic Identity Conference Proceedings, SeventhInternational Symposium on Human Identification; Jean-Francois Mercier,Gary W. Slater, and Pascal Mayer, Biophysical Journal Volume 85 Oct.2003 2075-2086). In this embodiment, the beads are then exposed togenomic DNA or mRNA and BRIDGE PCR(SAP) performed under conditions thatare designed to amplify specific nucleic acid sequences in the sampleincluding but not limited to entire genes or regions of genes. The beadswhich will have after the previous step amplified DNA attached to themare then placed in a cell-free protein synthesis system and the attachedDNA sequences used as templates for protein transcription andtranslation as described previously. One example of such a cell-freeprotein system is rabbit reticulocyte which is capable of supportingboth transcription and translation. A second system is a reconstitutedE. coli an example of which is the reconstituted system available fromPost Genome Institute Co., Ltd. (Japan) called PURESYSTEM. The systemswas originally developed at the University of Tokyo and comprisesapproximately 30 purified enzymes (enzymes made recombinantly) necessaryfor transcription and translation. Because all the components are taggedwith a hexahistidine, the preferred N-terminal and C-terminal epitopesfor the wild-type and truncated polypeptides (discussed in variousembodiments of the method below) are preferably not Histags. The systemis advertised as “essentially free of protease,” however, there issignificant protease activity that interferes with detection of smallpolypeptides by mass spectrometry. In one embodiment, the presentinvention contemplates supplementing a reconstituted system with aprotease inhibitor.

In order to capture the translated protein on the same bead as thetemplate nucleic acid produced using BRIDGE amplification, an affinitycoupling agents is utilized which can be attached to the bead surfaceand in addition may include the bead interior. Since some couplingagents such as ordinary proteins will denature under high temperatureconditions which might be encountered during the BRIDGE amplificationstep, a variety of coupling agent which are not damaged by the hightemperature conditions can be utilized. One example of a coupling agentwhich will not lose its native affinity after being heated to hightemperature even above 100 C are nucleic acid aptamers describedpreviously. Such aptamers will unfold at high temperature but refoldwhen the temperature is lowered, thereby preserving the native affinityand high selectivity of the aptamer for specific target biomolecules. Inthe case of translated proteins, a common epitope tag can be added bymodification of the primers which is recognized by the aptamer with highaffinity. Additional example of coupling agents which are compatiblewith BRIDGE amplification are single domain antibodies sometimesreferred to as nanobodies. Such single domain antibodies displaystability at much higher temperatures than ordinary antibodies. Anadditional example is of a affinity agents possessing stability totemperature are certain chelating agents such as Ni²⁺ which are attachedto the bead surface and display an affinity to so-called histidine tagsconsisting of several histidine residues positioned at either the N orC-terminal end of a protein. In all of these examples, coupling agentscan be directed to a specific epitope which is produced duringtranslation of the protein, thereby providing a means for binding of thetranslated protein to the same beads which have attached the nucleicacid sequences coding for said translated protein.

Coupling agents can also be attached to beads after BRIDGE amplificationis performed in order to avoid damage to the coupling agent that mightoccur due to high temperatures. For example, a variety of methodswell-known in the literature exist for attaching coupling agents tobeads. Since, in most applications the same coupling agent is used forall beads, the coupling agent can be attached in one step to the beadssubsequent to BRIDGE amplification. In one preferred embodiment, biotinis used with a chemically active moieties forms covalent bonds withamino group on a bead surface. After BRIDGE, a streptavidin conjugatedthrough photocleavable biotin to an antibody directed at a specificepitope is added to the bead population under conditions such that thestreptavidin interacts with the biotin preattached to the bead surface.This method provides a convenient method to create a photocleavablelinker between the antibody and the bead surface after BRIDGEamplification.

One preferred embodiment of this invention is directed to a method forconversion of a cDNA library to a complete or partial protein beadlibrary such that different proteins or polypeptides which are coded byelements of the cDNA library are attached to individual beads. Thisso-called bead sorted library of in vitro expressed proteins(BS-LIVE-PRO) is produced by providing a cDNA library and a set ofbeads, said beads each containing a set of forward and reverse primersdesigned to amplify using BRIDGE specific elements of the cDNA library.Each beads also has attached an affinity coupling element which exhibitsstability to high temperature up to 100 C and is directed against acommon epitope which is coded for by at least one element each primerpair attached to beads. The cDNA and beads are then introduced to thebeads and solid phase polymerase amplification (SPA) performed. Thebeads are subsequently introduced into a cell-free protein synthesissystem and coupled transcription and translation performed underconditions suitable to produce proteins which become attached throughthe said primer coded epitope to the said affinity coupling agentattached to the bead.

In one preferred embodiment, the proteins that are captured onto thebead surface using the methods described herein can be photo-released.This can be accomplished by using a coupling agent such as a singledomain antibody which is attached to the bead through a photocleavablelinker. It is important for the creation of a bead sorted proteinlibrary that this photocleavable linker does not lose its propertiesduring SPA. One example is the utilization of beads coated withstreptavidin which bind a photocleavable biotin attached photoreversiblyto a high temperature stable coupling agent such as described previouslyin this invention. One example is the utilization ofstreptavidin-photocleavable biotin linkage. It has been shown thatstreptavidin-biotin complexes exhibit unusual thermal stability up to117° C. (Gonzalez, M., Argarana, C. E., and Fidelio, G. D. (1999) BiomolEng 16, 67-72). The ability to photocleavable release of the translatedproteins captured on a bead surface is especially useful for directcontact photo-transfer of said proteins to a surface for subsequentanalysis and utilization in biomolecular detection applications.

Application to the Multiplex Detection of Mutations in Genes

One preferred embodiment of this invention is directed to the multiplexdetection of mutations in one or more genes such as part of a clinicaldiagnostic assay. A library of beads each containing specific proteinsor fragments of proteins is prepared from a sample of genomic DNA usingthe methods described in this invention including BRIDGE amplificationof specific genes or regions of genes on individual beads, cell-freetranslation of proteins or polypeptides coded for by the BRIDGEamplified DNA on separate beads and capture of proteins on beads whichattach the coding DNA through a photocleavable coupling agent. Theproteins or polypeptides on the bead are then transferred to a surfacethrough the method of direct photocleavage contact printing describedpreviously in this invention.

In one embodiment, the proteins are transferred to a surface suitable toperform MALDI analysis as previously described. It is to be understoodthat since each bead contains a homogeneous population of protein orpolypeptide which was coded for by the BRIDGE amplified nucleic acidalso attached to the bead, direct photocleavage contact printing asdescribed previously will produce a plurality of spots on the surface,each spot containing a distinct species of protein or polypeptide. Theidentity of the protein or polypeptide as well as the presence ofmutations can then be determined by measuring the molecular weight ofthe proteins/polypeptides as well as any possible shift in molecularweight caused by a mutation. The presence of a peak in the mass spectrumdue to the unaltered wild-type species not containing the mutation isassured as long as the mutation appears in only one of two chromosomespresent in all cases of heterozygous mutations.

In one preferred embodiment directed at the detection of APC mutationswhich are associated with both inherited and sporadic forms ofcolorectal cancer, a sample of genomic DNA derived from either blood orstool is provided. Specific regions of the APC gene are amplified usingBRIDGE amplification methods whereby primer pairs are provided on beadswhich are designed to amplify specific regions of the APC gene to bescanned for mutations. Primer pairs also incorporate sequences forpromoters for efficient transcription of the coded proteins as well as avariety of sequences encoding one or more epitopes for capture andanalysis of the protein. After BRIDGE amplification, the beads areincorporated in a cell-free protein synthesis system suitable fortranslation of the encoded proteins/polypeptides. Due to the selectivecapture of proteins on beads containing the coding DNA, each bead willcontain a homogeneous population (or nearly homogeneous, i.e. at least90% identical with 10% or less: contaminating, more preferably at least95% identical with 5% or less contaminating, still more preferably atleast 99% identical with 1% or less contaminating) ofproteins/polypeptides which can then be transferred by photo-release ofthe proteins to a MALDI surface using the methods described in thisinvention. By analyzing each transferred spot separately by MALDI,mutations in specific regions of the APC gene can be detected.

Application to the High Sensitivity Detection of Mutations

One further embodiment of the present invention applies to the detectionof chain truncation mutations which are known to be associated with avariety of genetically related diseases including but not limited tocancers such as colorectal, lung and breast cancer. Very often mutatedgenes that are either inherited or produced somatically in individualcells can trigger cancer either alone or in conjunction with othercauses such as additional mutations. The detection of such mutations,especially when they are present at very low concentration in abiological sample, relative to the wild-type gene sequence (unmutatedgene) is an important challenge and goal in biotechnology. This isespecially true in the case of colorectal cancer, where the detection ofchain truncating mutations in the APC gene is correlated with thepresence of polyps, precancerous adenomas or cancerous tumors in thecolon.

One embodiment of the present invention facilitates the detection ofcancer by creating a bead sorted library of in vitro expressed proteinsor polypeptides (BS-LIVE-PRO) from a patient sample containing DNA.Patient samples can include but are not limited to urine, stool, tumortissue, saliva, buccal scrapes or washes, cerebrospinal fluid orsynovial fluid. The said BS-LIVE-PRO are created from the patient sampleDNA using methods described in this invention such that each beadcontains either predominantly full-length (untruncated peptides)reflecting the presence of a wild-type sequence of a target gene orpredominantly truncated peptides reflecting the presence of a mutantsequence causing a chain-truncation. The beads are then probed for thepresence of the full-length or truncated protein using a variety ofassays.

One such assay which is highly advantageous for this application issimilar to the ELISA protein truncation test (ELISA-PTT) reported byGite et al. in 2003 [Gite et al. (2003) Nat Biotechnol 21, 194-197].This test can be configured to utilize fluorescently labeled antibodiesto probe the C- and N-terminal portions of the peptides bound toindividual beads.

In one preferred embodiment of this invention directed at detecting withhigh sensitivity chain truncating mutations occurring anywhere in a geneor portion of a gene the following is provided: i) a patient samplecontaining DNA and ii) beads which contain at least one of a set offorward and reverse primers designed to amplify a specific geneticsequence contained in said patient DNA and an affinity coupling elementwhich is directed against a nascent protein expressed from the sequencewhich is amplified by said primers. Alternatively, specific chemicalmoieties are present on the beads before amplification or created on thebeads during amplification and used to attach the affinity couplingelement after amplification, whereby the affinity coupling agent isdirected against a nascent protein expressed from the sequence which isamplified by said primers. The DNA from the patient sample (patient DNA)is added to the said beads and polymerase amplification is performedunder conditions such that the surface attached amplicon on said bead isderived from a few copies of patient DNA (preferably 10 but more optimal3, and even more optimal 1). The beads are subsequently introduced intoa cell-free protein synthesis system and coupled transcription andtranslation performed under conditions suitable to produce nascentproteins which become attached through the affinity coupling agent tothe bead. The nascent proteins on individual beads are then probed todetermine the presence or absence of truncated polypeptides. The ratioof beads with detected chain truncated polypeptide to those where suchchain truncated polypeptide is not detected is used to determine thefraction of patient DNA containing chain truncating mutations in thetargeted genetic sequence.

A variety of methods can be utilized to capture DNA on individual beadswhich are derived from a single copy chain reactions. One such methodutilizes solid phase polymerase amplification (SPA) and BRIDGE asdescribed previously. In this case, the concentration of DNA from thepatient sample is diluted sufficiently so that the solid phasepolymerase amplification on each bead is initiated by a single templateusing primer pairs that are immobilized on the bead surface. A secondapproach (e.g [Dressman et al. (2003) Proc Natl Acad Sci USA 100,8817-8822]), utilizes emulsions which trap single copies of the sampleDNA for subsequent amplification and immobilization of the product onthe bead surface. In either case, the amplified DNA can then be utilizedin a coupled cell-free transcription/translation reaction to expressnascent proteins ultimately derived from the product of the single copyPCR reaction.

Once a BS-LIVE-PRO is produced using the methods described above, thedetection of beads containing predominantly chain-truncated polypeptidescan be detected using a variety of methods. In one embodiment [Gite etal. (2003) Nat Biotechnol 21, 194-197] described in U.S. Pat. No.7,101,662 which is specifically incorporated by reference, two differentantibodies are used which are directed towards the N- and C-terminalportions of the expressed nascent protein. The binding of bothantibodies indicates a full-length peptide whereas binding of only theN-terminal directed antibody indicates a truncated peptide. Binding ofthe antibodies can be detected using a variety of different methodsincluding a fluorescent or chemiluminescent read-out. For example, duelor single labeled nascent proteins bound to individual beads can bedetected using a sensitive microarray scanner or with flow cytometry.

Even if only a small proportion of the DNA in the patient sample encodesfor a chain truncated polypeptide, these should be detectable by probingthe individual beads. For example, if 1 out of 100 copies of DNA encodedfor a gene contain a chain truncating mutations, approximately 1 out of100 beads should contain predominantly polypeptides which were altereddue to the chain truncating mutation. Importantly, this approach allowsrapid scanning for chain truncating mutations in an entire sequence of agene without pre-knowledge of the mutation in contrast with reportedmethods which are designed to detect specific mutations or singlenucleotide polymorphisms (SNPs) at the DNA level [Dressman et al. (2003)Proc Natl Acad Sci USA 100, 8817-8822; Diehl et al. (2005) Proc NatlAcad Sci USA 102, 16368-16373].

The methods described in this invention can also be utilized to transferthe nascent proteins from individual beads onto discrete spots on thesurface by means of phototransfer. In this case, each individual spotcan be probed to determine if it contains predominantly truncated orfull-length peptide or protein.

One preferred method of determining if a photo-transferred spot on asurface contains a predominantly truncated or full-length peptide is theuse of mass-spectrometry and more preferably MALDI mass spectrometry asdescribed previously in this invention. In this case, a shift in mass ofthe polypeptide from that predicted for the WT sequence would indicatethe presence of a mutation.

It will be understood by those skilled in the use of mass spectrometryto probe proteins and polypeptides that many mass spectrometer whichhave high sensitivity and high mass resolution would allow not onlychain truncation mutations to be detected but any mutation whichresulted in a shift in the mass of the expressed peptide. For example,many commercially available MALDI mass spectrometers such as the ABI4800 have sensitivity sufficient to detect mass shifts of much less than1 dalton. Thus, beads which have bound predominantly nascent proteinexpressed from the normal wild-type sequence of a gene will produceeasily distinguished signal from those beads which have boundpredominantly mutant protein expressed from a mutant sequence providedthat the mutation produced a peptide with a mass shift of at least 1dalton. It will be also recognized by those skilled in the art of massspectrometry of proteins and polypeptides that in many cases the actualchange in the amino acid sequence of the polypeptide can be determinedby utilizing peptide sequencing capabilities of many commerciallyavailable mass spectrometers such as the ABI 4800.

In one preferred embodiment of this invention directed at detecting atscanning with high sensitivity mutations occurring anywhere in a gene orportion of a gene the following is provided: i) a patient samplecontaining DNA and ii) beads which contain at least one of a set offorward and reverse primers designed to amplify a specific geneticsequence contained in said patient DNA and an affinity coupling elementwhich is directed against the nascent protein expressed from thesequence which is amplified by said primers. The DNA from the patientsample (patient DNA) is added to the said beads and polymeraseamplification is performed under conditions such that the surfaceattached amplicon on said bead is derived from a few copies of patientDNA (preferably 10 but more optimally, 3 and even more optimally 1). Thebeads are subsequently introduced into a cell-free protein synthesissystem and coupled transcription and translation performed underconditions suitable to produce nascent proteins which become attachedthrough the affinity coupling agent to the bead. The nascent proteins onindividual beads are then probed to determine the presence or absence ofmutant polypeptides. The ratio of beads with detected mutant polypeptideto those where such mutant polypeptide is not detected is used todetermine the fraction of patient DNA containing mutations in thetargeted genetic sequence.

In addition to mass spectrometry a variety of methods exist to assay thenascent protein derived from each bead. This includes assaying nascentprotein bound directly to a bead or photo-transferred to a surface.Useful methods well know to those skilled in the area of proteinanalysis, biotechnology and biophysics include but are not limited tousing fluorescence, chemiluminescence, absorption, Raman spectroscopy,infrared spectroscopy, mass spectrometry, flow cytometry, multiphotonspectroscopy, multiphoton microscopy, single molecule detection,functional analysis and microarray analysis. For example, as describedpreviously proteins nascent proteins which have altered sequences canoften be detected by mass spectrometry provided the mass of the alteredsequence is not degenerate with the wild-type sequence. In the case ofchain truncated polypeptides fluorescent labeled antibodies orantibodies which have a chemiluminescent readout can be utilized toprobe the relative proportion of the N-terminal and C-terminal ends ofthe nascent protein. In some cases, the nascent protein can be probedfor functional activity which is disrupted by changes in the wild typesequence. For example, it is well known that many mutations will alterthe binding property of p53 for specific sequences of DNA. Raman andinfrared spectroscopy can be used to detect changes in the overallstructure and amino composition of proteins and polypeptides.Multiphoton spectroscopy and multiphoton microscopy can provide a meansto probe with high spatial resolution the presence of specificchromophores which might be present or interacted with a nascent proteinand with long wavelength non-damaging light.

Application to a Protein Truncation Test

One preferred embodiment of this invention is directed to the detectionof chain truncating mutations in genes using methods described in thisinvention. Chain truncating mutations which result in truncated geneproduct, are prevalent in a variety of disease-related genes [Den Dunnen& Van Ommen. (1999) Hum Mutat 14, 95-102], including APC (colorectalcancer) [Powell et al. (1993) N Engl J Med 329, 1982-1987; van der Luijtet al. (1994) Genomics 20, 1-4; Traverso et al. (2002) N Engl J Med346,311-320; Kinzler et al. (1991) Science 251, 1366-1370; Groden et al.(1991) Cell 66, 589-600.], BRCA1 and BRCA2 (breast and ovarian cancer)[Hogervorst et al. (1995) Nat Genet. 10, 208-212; Garvin. (1998) Eur JHum Genet. 6, 226-234; Futreal et al. (1994) Science 266, 120-122.],PKD1 (polycystic kidney disease) [Peral et al. (1997) Am JHum Genet. 60,1399-1410.], NF1 and NF2 (neurofibromatosis) [Heim et al. (1995) Hum MolGenet. 4, 975-981; Parry et al. (1996) Am J Hum Genet. 59, 529-539.] andDMD (Duchenne muscular dystrophy) [Roest et al. (1993) NeuromusculDisord 3, 391-394.]. Such chain truncating mutations can be detectedusing the protein truncation test (PTT), well known in the diagnosticfiled. However, this test is based on cell-freetranscription/translation of PCR(RT-PCR) amplified portions of thetarget gene (or target mRNA) followed by analysis of the translatedproduct(s) for shortened polypeptide fragments. However, conventionalPTT is not easily adaptable to high-throughput applications since itinvolves SDS-PAGE followed by autoradiography or Western blot. It isalso subject to human error since it relies on visual inspection todetect mobility-shifted bands.

To overcome these limitations, a solid-phase PTT (so-called ELISA-PTT)was developed [Gite et al. (2003) Nat Biotechnol 21, 194-197]. Oneembodiment of ELISA-PTT uses a combination of misaminoacylated tRNAs[Rothschild & Gite. (1999) Curr Opin Biotechnol 10, 64-70; Gite et al.(2000) Anal Biochem 279, 218-225.], which incorporate affinity tags forsurface capture of the cell-free expressed protein fragments, andspecially designed PCR primers, which introduce N- and C-terminalmarkers for measuring the relative level of shortened polypeptideproduced by the chain truncation mutation. After cell-free translationof the protein fragments, capture and detection is accomplished in asingle-well using a standard 96-well microtiter plate ELISA format andchemiluminescence readout. The technique was demonstrated for thedetection of chain truncation mutations in the APC gene using DNA or RNAfrom cancer cell lines as well as DNA of individuals pre-diagnosed withfamilial adenomatous polyposis (FAP) [Gite et al. (2003) Nat Biotechnol21, 194-197].

A second version of this approach uses three epitopes described in U.S.Pat. No. 7,101,662 which is specifically incorporated by reference. Inthis approach, two epitopes located near the N-terminal end of thecell-free expressed protein or protein fragment are incorporated using aspecially designed forward primer during PCR. These epitopes serve thepurpose of binding the expressed protein or protein fragment to asurface and detection of the N-terminus. A third epitope tag,incorporated at the C-terminal end of the protein or protein fragment,by the reverse primer during PCR, is used for detection of theC-terminal end which is absent in the case of chain truncationmutations.

In the case of the present embodiment regarding a bead-based PTT, amethod is used comprising: a) providing i) a population of templatemolecules, each template molecule encoding a nascent protein or proteinfragment, and ii) at least one surface comprising forward and reversePCR primers attached to said surface; b) amplifying at least a portionof said population of template molecules so as to create amplifiedproduct attached to said surface; c) generating nascent protein orprotein fragment from said amplified product, said nascent protein orprotein fragment comprising an affinity tag or first epitope, anN-terminal detection tag or second epitope and a C-terminal detectiontag or third epitope; d) capturing said nascent protein or proteinfragment on said surface via a first ligand, said first ligand attachedto said bead and reactive with said affinity tag or first epitope; e)detecting N-terminal end of said nascent protein or protein fragmentsvia a second ligand, said second ligand attached to a detection moiety;and f) detecting C-terminal end of said nascent protein or proteinfragment via a third ligand, said third ligand attached to a detectionmoiety.

In one embodiment, the template molecules are derived from genomic DNAor fragmented genomic DNA that are present in common patient samplesincluding but not limited to blood, plasma, serum, urine, sputum,saliva, stool, mouth lavage and buccal scrape/swab. The affinity bindingtag consists of an HSV epitope sequence, the N-terminal detection tag aVSV epitope sequence and the C-terminal detection tag a p53 epitopesequence. The cell-free expressed protein or protein fragment iscaptured on a bead surface using an antibody directed against the HSVepitope. In order to distinguish between full-length and truncatedpolypeptides on a bead, two fluorescently labeled antibody ligandsdirected against the VSV and p53 epitopes are employed, each with adifferent wavelength of fluorescence emission which can be detectedseparately without significant wavelength overlap. For example, thecombination of red and green fluorescence from the N-terminal andC-terminal antibodies indicates a full-length peptide whereas only greenfluorescence indicates a truncated polypeptide. As described in thisinvention, individual beads can be read using a microarray scanner,fluorescence microscope or flow cytometer.

In one embodiment, the second ligand directed against the N-terminalepitope (second epitope) and third ligand directed against theC-terminal epitope (third epitope) are labeled with detection moietieswhich are chosen to act as donor and acceptor pairs, for fluorescenceresonance energy transfer (FRET). For example, if the detection moietyon the second ligand is a donor and the detection moiety on the thirdligand an acceptor, then the fluorescence from the donor will bequenched when excited at the wavelength of maximum excitation as long asthe donor/acceptor pair are close to each other (e.g. within 100Angstroms and more preferably within 50 Angstroms). In this case, onlythe acceptor will fluoresce at its characteristic wavelength normallyred-shifted from the donor fluorescence or if it is a “dark” quencher[Johansson et al. (2004) J Am Chem Soc 126, 16451-16455], it will quenchthe donor but not itself fluoresce. It will be readily understood bythose skilled in the art that the use of FRET detection pairs asdescribed above enables preferential detection of chain truncatedpeptides from full-length peptides since the N-terminal ligand labeledwith a donor will only fluoresce when the C-terminal ligand with theacceptor moiety is not present.

In order to demonstrate the process of bead-based fluorescence PTT, atest assay was designed using PCR amplification of segments of the APCgene from cell-line genomic DNA. The corresponding polypeptide wasexpressed in a rabbit reticulocyte cell-free transcription/translationsystem (RRL) and captured on 100 micron diameter NeutrAvidin coatedagarose beads, which were loaded with a capture antibody. Note that thecapture antibody was bound to the NeutrAvidin coated agarose beadsthrough AmberGen's proprietary photocleavable biotin, to facilitatephoto-release or contact photo-transfer in cases where desired. The PCRprimers were designed to amplify APC segment 3 of Exon 15, whichcorresponds to codons 1,099 to 1,696. In addition to the wild-type (WT)sequence (HeLa cell line), cell-line genomic DNA containing a chaintruncation mutation at codon 1,338 of APC(CAg→TAg) was used as thetemplate for PCR(SW480 cell line). Similar to the ELISA-PTT assay, threeepitope tags were incorporated into the PCR amplified DNA via thespecially designed primers. These included an HSV epitope tag forbinding to the corresponding antibody on the beads, a VSV epitope tagfor N-terminal readout and AmberGen's proprietary p53 epitope tag forC-terminal readout. Exploiting the photocleavable biotin linkage of thebinding (capture) antibody, APC polypeptides were contactphoto-transferred from the beads to a microarray substrate prior todetection with the fluorescence antibodies.

In one embodiment, the present invention contemplates 2-colorfluorescence overlays which show N-terminal and C-terminal detection(for example, in one embodiment, green is the anti-VSV-Cy3 N-terminaldetection and red is the anti-p53-Cy5 C-terminal detection, with yellowbeing the combination of both colors). The minus DNA negative controlsample (no DNA during cell-free protein expression) shows zero signaldue to the use of contact photo-transfer, which eliminatesauto-fluorescence arising from the beads themselves as well asfluorescence on the beads due to non-specific binding (e.g. of thedetection antibodies). Importantly, the chain-truncating mutant displaysonly the VSV signal (green) while the WT has both VSV and p53 (red andgreen which appears yellow in the overlay). Intrinsic fluorescentlabeling of the APC polypeptide (both full-length and truncated) usingFluoroTect tRNA labeling was also detected confirming that polypeptidewas bound to bead independent of N-terminal measurement. Details of thisexperiment are described in Example 42 of the Experimental section.

Bead-Based PCR Amplification of DNA Combined with Polypeptide Cell-freeExpression

An additional embodiment of this invention is the production of beadscoated with polypeptide from beads coated with specific primers plustemplates coding for a specific gene or gene fragment. In one example,customized primers attached to beads (both forward and reverse) are usedto capture target DNA through hybridization (step 1). These primers aredesigned to amplify specific regions of a particular gene, for example,a portion of the DNA coding for the APC gene, as well as incorporate the3 epitope tags and comprise additional sequences which promote cell-freetranslation (optimized for specific cell-free reaction systems such asE. coli or rabbit reticulocyte). Beads are also coated with an affinityagent, such as biotin, which is used for attachment of the captureantibody later in the process. After hybridization-capture of the targetDNA directly on the bead (e.g. fecal DNA isolated from stool samples oralternatively freely circulating DNA present in other assay samples suchblood or urine) the DNA may be separated from non-hybridizing DNA byremoving the beads from the assay solution followed by a washing step.The target DNA captured on each bead is then selectively amplified usingthe BRIDGE PCR process (step 2), thereby yielding beads coated withtemplate DNA coding for the desired polypeptide sequence to be probed. Acapture antibody is then attached to the beads (step 3) through a(strept)avidin-biotin interaction (using the tetrameric (strept)avidinas a bridge between biotin on the beads and biotin on the captureantibody). Note that the antibody may optionally be connected throughphotocleavable biotin described in this invention for the purpose ofcontact photo-transfer. This DNA is then transcribed/translated, forexample in some cases in an ultra-low protease protein expressionsystem, and the polypeptide subsequently captured on the same bead fromwhich it was made. Capture is achieved via the capture antibody on thebeads and the incorporated N-terminal epitope in the expressed proteins.

An important feature of the method of this embodiment is the ability toperform multiplexed solid-phase PCR (SP-PCR) (e.g. BRIDGE) reactionsfollowed by multiplex cell-free protein expression reactions. Sincemixing of the resulting proteins from a particular bead (parent bead) toanother bead (non-parent bead) during this process is minimized, theexpressed proteins are essentially sorted on individual beads (on theirparent DNA coated beads). This is especially valuable for multiplexingof different segments of a gene (e.g. specific exons or otherfragments), for example in a bead-based PTT assay.

As an example of this process including a simple 2-fold multiplexing, anexperiment was performed which was designed to express two differentmodel proteins, p53 and γ-actin, separately on individual beads. Detailsof each step used including primer design, primer binding to beads,solid-phase PCR and the cell-free expression reaction are described inExample 31 of the Experimental section.

-   -   1) Primers: First, gene-specific primers were designed similar        to that used in a recent AmberGen publication [Gite et        al. (2003) Nat Biotechnol 21, 194-197] which included regulatory        sequences necessary to convert the DNA template to a form which        can be expressed in a rabbit reticulocyte cell-free system. The        overall sequences included a T7 promoter, a Kozak ribosome        binding sequence (forward primer) and an HSV epitope tag        (reverse primer).    -   2) Primer Attachment to Beads: Primers were purchased        commercially (Sigma-Genosys), each with 5′ amine modifications        for bead attachment. Both primers, along with a biotin-amine        linker (Biotin-PEO-Amine; Pierce Biotechnology), were then        covalently attached to ˜100 μm amine-reactive NHS ester        activated 4% agarose beads (Amersham Biosciences). The        co-attachment of biotin provides a heat stable molecular        “handle” for later attachment of (strept)avidin and        photocleavable biotin (PC-biotin) conjugated PC-antibodies.    -   3) BRIDGE PCR: After completing all covalent bead modifications        and extensive washing, successful primer and biotin attachment        was separately confirmed. Beads with different gene-specific        primer sets were then pooled and a single-tube SP-PCR reaction        was performed under standard PCR conditions (no soluble        primers). An in-house prepared cDNA library was used as the PCR        template.    -   4) Adding the Capture Antibody: A PC-biotin conjugated        PC-antibody against the common HSV epitope tag was bulk loaded        onto the beads using a NeutrAvidin bridge. Successful loading        was confirmed using a secondary detection antibody.    -   5) Protein Expression and Microarray Printing: Fully prepared        DNA-beads were then cell-free expressed using        BODIPY-FL-tRNA^(COMPLETE) (TRAMPE) to label all nascent protein        (green). Following contact photo-transfer of the beads to an        epoxy activated microarray slide, the microarray was then probed        with the p53 antibody clone Bp53-12 (B-P3) (BioSource        International) which was in-house labeled with Cy5 fluorescence        (red) (Amersham Biosciences).        Bead-Based Digital PCR without Limiting Dilution or        Encapsulation

Digital PCR, especially when applied to a bead format, is an importantadvance in biotechnology. It facilitates a variety of applicationsincluding massively parallel DNA sequencing, for example of genomes[Dressman et al. (2003) Proc Natl Acad Sci USA 100, 8817-8822; Kojima etal. (2005) Nucleic Acids Res 33, e150; Nakano et al. (2003) J Biotechnol102, 117-124; Nakano et al. (2005) J Biosci Bioeng 99, 293-295; Shendureet al. (2005) Science 309, 1728-1732; Thomas et al. (2006) Nat Med 12,852-855]. This relies on the ability to amplify single copies or a mosta few copies of template DNA on a single bead. However, single copy PCRhas only been demonstrated up to now using an emulsion method wherebybeads are trapped in an emulsion with approximately one molecule of DNAby using limiting dilution; e.g. diluting the concentration of thesolution so that the average number of molecules encapsulated along witha single bead is one [Dressman et al. (2003) Proc Natl Acad Sci USA 100,8817-8822].

One embodiment of this invention is directed at the performance ofbead-based digital PCR without the need for limiting dilution orencapsulation of the bead. Such an approach avoids many of thelimitations of conventional bead-based digital PCR, for example:

-   -   1. Limiting dilution requires careful adjustment of the DNA at        very low concentration, a process difficult and expensive to        automate.    -   2. The bead encapsulation introduces extra steps in any assay        resulting in higher cost.    -   3. The small 1 micron beads used in conjunction with bead        encapsulation are difficult to read with a standard microarray        scanners with a resolution greater than 3 microns.    -   4. With regards to digital PTT, in order to perform cell-free        transcription/translation, the bead encapsulation needs to be        removed in order to allow large macromolecules such as        polymerases and ribosomes to have access to the bound template.

Although embodiments of this invention are directed at molecular assaysperformed at the protein level, such as bead-based digital PTT, it willbe realized by those skilled in the field that this embodimentfacilitates a variety of other useful applications at the DNA levelincluding bead-based massively parallel DNA sequencing or SNP/mutationanalysis, for example by single-base extension.

One very desirable feature of this embodiment is the ability to performamplification of DNA and subsequent production of proteins (e.g.polypeptides) without encapsulation methods. This is possible because:i) the PCR amplification is confined to the solid-phase on individualbeads due to the intrinsic nature of the BRIDGE process. This limits thepossibility that amplicon escapes from the bead and binds to other beadsin the vicinity of the local reaction. ii) The transcription/translationreaction occurs at or near the surface of the bead since the templateDNA is covalently attached to the bead surface. iii) Capture antibodiesdirected at an affinity tag on the translated polypeptide act to capturethe said polypeptide before it escapes from the bead thereby minimizingmixing with other beads. Together these factors ensure that even withoutencapsulation, the overall PCR amplification and subsequent polypeptidetranslation is confined to the bead.

Another advantageous feature of this embodiment is the ability toperform amplification on a few copies or ideally a single copy of atemplate DNA on a single bead despite the fact that the template to beadratio in solution is initially much higher than a 1:1 ratio.

The ability to obtain a higher ratio relates to the ability of thetemplate DNA to hybridize to the covalently bound primer on the beadsurface. Under certain well defined conditions, this requires a muchhigher ratio of template to bead than the normal 1:1 conditions used forbead encapsulation (e.g. 5:1 or preferably higher than 10:1). Forexample, the binding of single copies of target DNA to the bead dependson a number of factors including melting temperature of the primermolecules and target DNA template, the relative net charge andhydrophobicity of the bead, as well as in the case of agarose beads theproperties of the intrinsic polymer matrix which both limit the abilityof the target DNA to penetrate into the bead and hybridize with theprimers. These factors can be controlled for example by adjusting theagarose density, hybridization temperature and melting temperature ofthe template DNA-primer.

An additional useful step in this embodiment is the removal of excesstemplate copies in the solution bathing the beads, prior to performingadditional PCR amplification cycles. For this purpose, after initialhybridization-based capture of a few copies of the template on the beadsurface and a subsequent first cycle of BRIDGE PCR amplification (i.e.extend the primers only once), the initial non-covalently attachedtemplate is then removed from the bead (leaving only the covalentlyattached primer extension products, i.e. PCR products); assuring thatthe subsequent solid-phase BRIDGE PCR reaction (additional cycles) isconfined to the bead surface and does not involve additional templates,as in the case of the conventional bead-based PCR reaction which occurspartially in the solution phase.

The overall method comprises of several steps:

-   -   1) A few (preferably one) target DNA molecules are captured on a        single bead through hybridization with specially designed        primers which hybridize with regions of the DNA which is to be        analyzed (e.g. for chain truncation mutations).    -   2) A single cycle of PCR is performed so that each captured copy        of target DNA serves as a template to extend the primer which is        covalently linked to the bead surface.    -   3) All non-covalently bound copies of the target DNA are        de-hybridized for example by denaturation in NaOH and removed by        washing to prevent further “seeding” of the bead (2^(nd) panel).    -   4) Additional cycles of the BRIDGE PCR amplification reaction        are then performed in a PCR solution devoid of additional        template (bottom two panels show only two cycles).

Application to Massively Parallel Sequencing Systems

BS-LIVE-PRO as produced using the methods described above can be usedand analyzed in conjunction with a new generation of bead basedmassively parallel DNA sequencing systems to provide many importantadvantages in the fields of proteomics and molecular diagnostics. Forexample, several instruments which are commercially available can beused in their existing form or with some modification for proteomic anddiagnostic applications due to the unique features of BS-LIVE-PRO andthe methods engendered by this invention.

For example, the Genome Sequencer 20™ System, developed by 454 LifeSciences can be used in conjunction with BS-LIVE-PRO for both proteomicand molecular diagnostic applications. This system is anultra-high-throughput automated DNA sequencing system capable ofresolving hundreds of thousands of DNA sequences in one run. The basicchemistry utilizes the release of pyrophosphate (PPi) that occurs witheach nucleotide addition during DNA-directed DNA synthesis to generatean amount of light commensurate with the amount of PPi released; thislight is captured by a CCD camera and converted into a digital signal.The combination of signal intensity and positional information over thePicoTiterPlate™ device (see below) allows the Sequencer's Linux-basedcomputer, equipped with an onboard Field Programmable Gate Arrayprocessor, to determine the sequence of hundreds of thousands ofindividual reactions simultaneously, producing millions of nucleotidesof sequence per hour.

In one preferred embodiment of this invention which utilizes a beadbased massively parallel sequencing system such as the Genome Sequencer20™ System, a cDNA library is converted using the methods described inthis invention into a complete or partial protein bead library such thatdifferent proteins or polypeptides which are coded by elements of thecDNA library are both attached to individual beads. In other words eachbead contains the coding DNA and protein or polypeptide from which it isderived. This so-called bead sorted in vitro expressed protein library(BS-LIVE-PRO) is then analyzed using the capabilities of a bead basedmassively parallel sequencing system.

In one preferred embodiment of this invention which utilizes a beadbased massively parallel sequencing system, before the DNA residing oneach bead is sequenced, the protein which is coded for by the DNA isanalyzed for example to determine if a particular target protein ormolecule interact with any particular protein on specific beadscomprising the BS-LIVE-PRO. Once the proteins on the beads have beenanalyzed, the identity of the protein residing on the bead is thendetermined by sequencing the DNA residing on the bead. This normallyrequires sequencing of only a small portion of the actual DNA sequenceresiding on each bead in order to determine the identity of the protein.For example, for a 454 system only 100 base pairs and even morepreferentially 25 base pairs are only needed to establish the uniqueidentity of each protein residing on the bead.

There are a variety of means for which the beads can be analyzed andsubsequently sequenced which is compatible with the bead based approachfor massively parallel sequencers and with specific components typicallyincorporated in such systems. For example, many sequencers utilize beadsdeposited onto a surface or into preformed pits. Because the sequencersare designed to detect light originating from individual beads, thiscapability can be used to measure the interaction of the proteins on thebead with molecules which are directly or indirectly labeled with lightemitting substances such as fluorophores or chemiluminescent markers.Those skilled in this field will recognize that this capability derivesfrom the use of fiber optics where individual fibers collect light fromindividual beads or through the use of high resolution scanners whichare able to resolve the light being emitted from individual beads.

In one application, the beads comprising the BS-LIVE-PRO are exposed toa fluid sample containing a putative interactor such as a single proteinwhich may interact with one or more of the proteins residing on specificbeads or a more complex mixture such as serum from the blood of apatient which may contain antibodies which may interact with one or moreof the proteins residing on specific beads. Other examples includecandidate drug compounds which may interact with one or more of theproteins residing on specific beads. In each case, the beads which theputative interactors may interact with can easily be measured using theability of the sequencer instrument to measure light emitted fromindividual beads by attaching directly or indirectly a marker such as afluorophore or chemilumiscent molecule to the putative interactor Inparticular, once an interactor has bound to a particular bead, the lightemitted from the interactor is detected. This information plus thepositional information and sequence information from the individual beaduniquely identified the protein on the bead.

It will be understood by those familiar with DNA sequencers that it ispossible to perform many cycles of bead analysis using the processdescribed above. For example, a potential interactor which isfluorescently labeled can introduced into a chamber which encloses theDNA sequencer substrate where the beads will reside (e.g.PicoTiterPlate™ device in case of the Genome Sequencer 20™ System),washed out without displacing individual beads and then a secondfluorescently labeled interactor introduced in the chamber. This cyclecan be repeated multiple times and information determined about theposition of which beads interact with the fluorescently labeledinteractor followed by sequencing of the individual beads. In this way,the profile of how each protein residing on the beads interacts withmultiple interactors can be determined. It is also possible to usemultiple fluorophores which emit at different wavelengths to introducemore then a single interactor during each cycle.

In addition to bead based massively parallel DNA sequencing systems, anumber of parallel sequencing systems utilize non-bead technology basedon binding of single DNA molecules or islands of DNA derived from singleDNA molecules to substrates. Examples include the Solexa technology(Illumina Genome Analyzer) and the Helicos Biosystems, Inc. technology.The methods and compositions of this invention can be usedadvantageously with these non-bead based sequenicing systems. Forexample, in one preferred embodiment, a BS-LIVE-PRO is created and DNAand proteins are PC-printed onto a substrate which is subsequentlyanalyzed using the non-bead based sequencing methods.

In a second example, proteins are generated using methods described inthis invention directly from DNA randomly deposited onto the surface ofthe sequencing substrate as employed by both Helicos and Solexa and theproteins analyzed prior to performing DNA sequencing. In this case, thecombination of analysis of the proteins derived from the sequenced DNAprovides a unique advantages in terms of performing efficientlydiagnostic applications discussed above in conjunction with beads.

This contrasts for example, with methods reported previously of printingknown sequences of DNA on a surface followed by protein translation. Inthis case, DNA amplification must be performed for each species of DNAin separate PCR reactions prior to deposition on a surface. For anentire genome this might require as many as 20,000 separate PCRreactions. The methods presented in this invention avoid the need forsuch large number of reactions by using random deposition of singlemolecules of DNA on beads or surfaces followed by amplification and thentranslation to protein. Decoding is then performed using a massivelyparallel sequencing system.

DNA is immobilized on a proprietary flow cell surface designed topresent the DNA in a manner that facilitates access to enzymes whileensuring high stability of surface-bound template and low non-specificbinding of fluorescently labeled nucleotides. Solid phase amplificationis employed to create up to 1,000 identical copies of each singlemolecule in close proximity (diameter of one micron or less). Becausethis process does not involve photolithography, mechanical spotting orpositioning of beads into wells, Solexa sequencing technology canachieve densities of up to millions of single molecule clusters persquare centimeter.

Identification Tags in Primer Sequences for Simultaneous Analysis ofMultiple Patient DNA

The methods and compositions described in this invention can also beadvantageously applied to analyze mutations present in multiple patientsby introducing identification tags into primer(s) which are bound tobeads. Such identification tags comprise of a unique sequence of basesthat serve to code the origin of the template DNA from individualpatients which is amplified on the bead. By incorporating the uniqueidentifier sequence into primers, the amplified DNA which is covalentlybound to the surface of the bead as well as the expressed protein fromthat DNA can be uniquely identified with an individual patient eventhough beads carrying amplified DNA from multiple patients are pooled(e.g. mixed) together.

The number of bases used for the identifier sequence is determined bythe number of patients which will be simultaneously be analyzed. Forexample, 1000 patients can be uniquely identified by using only asequence of only 5 bases which yields 1064 unique sequence combinations.Additional bases might also be added in order to code additionalinformation such as the sample number from a particular patient wheremore then one sample has been collected, date, time and status ofpatient. Additional bases might also be added to the sequence in orderto provide a “check sum” which is determined using an algorithm based onthe prior sequence in order to test its validity.

One example would be the case of 1000 different patients where theprimer contains a sequence of 6 bases. The first 5 bases can uniquelydetermine which patients DNA has been amplified since there are 1064possible sequences using 4 different bases A=adenine, T=thymine,C=cytosine and G=guanine. The sixth base might be based on a simplealgorithm whereby each base is assigned a number A=1, T=2, C=3 and G=4.The numbers are summed and divided by 5 and rounded off to the nearestnone zero integer which determines the sixth base. Hence ATGGC=14 andwhen divided by 5 and rounded of to the nearest none zero integer is 3,thus the sixth base would also be a C. Thus skilled in the area ofcomputer science will recognize there are many possible algorithmspossible to develop check sums to increase the read reliability of thepatient tag identifiers.

Each patient sample containing DNA is amplified separately on beadsusing the methods described in this invention and then the resultingbeads containing the immobilized amplified DNA pooled together forsimultaneous analysis. Thus for example, in the case where beads areanalyzed for the presence of DNA coding for chain truncated peptides,subsequent sequencing of all beads using a preferred method such as amassively parallel DNA sequencer will reveal not only the segment of DNAwhere the mutation resides in the gene but also the identity of thepatient where the DNA template used for amplification of the DNA on thebead originated.

The use of identifier tags in primers immobilized on beads isparticularly advantageous in cases where massively parallel DNAsequencers are used to sequence the DNA on multiple beadssimultaneously. For example, many of the new generation of DNAsequencers can sequence simultaneously over 1 million beads however thenumber of beads necessary to analyze the DNA from an individual patientmay be far less (e.g. 1000 beads). The use of identification tags inprimer sequences provides a means whereby many patients (e.g. 1000) canbe simultaneously analyzed without the need to segregate the beads fromindividual into different sequencing compartments on the sequencer. Suchcompartmentalization requires segregation of beads at each step in thesequencing process including introduction of the beads associated witheach patient on the sequencing substrate (e.g. slide).

The use of identification tags in primers to code for individualpatients also does not require that the amplified DNA remain bound tothe individual bead. As described in this invention, DNA can betransferred directly to spots on a substrate from the beads usingPC-print methods described in this invention. For example, the DNA maybe amplified on the surface of a bead but analyzed on a surface otherthan the bead provided that the DNA from each individual bead remains inseparate deposited spots.

Identification tags in primers can also be used advantageously inconjunction with DNA sequencing methods that do not employ beads, yetstill capable of analyzing in parallel millions of individual DNAtemplates as for example employed by Solexa or Helicos Bioscience, Inc.In the case of Solexa, individual DNA templates are amplified by surfacePCR directly on a substrate to produce a series of individual islands ofDNA which are derived from a single template. These islands are thensequenced in parallel. For the purpose of analyzing specific regions ofmultiple patients DNA for particular mutations sequence identificationtags incorporated into primers can again be used to amplify the specificregion of a patients DNA which one wishes to sequence. In this case,solution PCR is performed in separate reactions for each patient DNA andthen the resulting amplified DNA can be pooled and applied to thesequencing system. In the case of Helicos Bioscience, Inc. singlestrands of DNA are sequenced, however, identification tags can still beemployed at the stage where specific regions of a patients DNA isamplified.

DEFINITIONS

The terms “bead”, “sphere”, “microbead” and “microsphere” are usedinterchangeably herein. Polymeric microspheres or beads can be preparedfrom a variety of different polymers, including but not limited topolystyrene, cross-linked polystyrene, polyacrylic, polylactic acid,polyglycolic acid, poly(lactide coglycolide), polyanhydrides,poly(methyl methacrylate), poly(ethylene-co-vinyl acetate),polysiloxanes, polymeric silica, latexes, dextran polymers and epoxies.The materials have a variety of different properties with regard toswelling and porosity, which are well understood in the art. Preferably,the beads are in the size range of approximately 10 nm to 1 mm, and canbe manipulated using normal solution techniques when suspended in asolution. Beads may be porous or non-porous. In some embodiments whereporous beads are employed, ligands may be attached within the bead aswell as on the bead.

Terms such as “connected,” “attached,” “linked,” and “conjugated” areused interchangeably herein and encompass direct as well as indirectconnection, attachment, linkage or conjugation unless the contextclearly dictates otherwise. In one embodiment, the present inventioncontemplates bead-ligand-nascent protein conjugates or complexes. Theattachment of the ligand to the bead may be covalent, while theattachment of the ligand to the nascent protein may be non-covalent. Ina preferred embodiment, compounds, ligands, etc. are covalently attachedto beads through a photocleavable linker. However, in some embodiments,there may be additional functional groups at one or more sites along thelinker.

As used herein, “binding agents” can be of any type. In one embodiment,the can be comprise chemical moieties. In a preferred embodiment,binding agents are ligands, such as antibodies, lectins, aptamers,streptavidin and avidin (and the like).

A “portion” can be with reference to a population or a molecule (e.g. agene), depending on the context. For example, where contacting resultsin at least a “portion” of said nucleic acid annealing to said one ormore amplification primers, it should be clear that portion is withreference to a population. Similarly, where at least a “portion” of saidprimers is extended, the term is with reference to a population.Similarly, when transferring at least a portion of said nascent proteinto a non-bead solid support, the term is with reference to a population.By contrast, a portion of a disease-related gene (“encoded by a portionof the APC gene”) is a region (e.g. larger than 4 bases, typically 8-15bases or more, preferably 20 bases or more).

As used herein, “bisulfite-treated” means exposure to a bisulfitecontaining reagent. Typically, bisulfite is used as an aqueous solutionof a bisulfite salt (e.g. sodium bisulfite, sodium metabisulphite). Ithas been discovered that bisulfite methods that employ magnesiumbisulfite, polyamine compounds, and/or quaternary amine compoundsprovide useful alternatives to sodium bisulfite conversion reactions.See “Method And Materials For Polyamine Catalyzed Bisulfite ConversionOf Cytosine To Uracil” (U.S. application Ser. No. 60/499,113 filed Aug.29, 2003, and also application Ser. No. 60/520,942 having the same titleand filed Nov. 17, 2003), “Method And Materials For Quaternary AmineCatalyzed Bisulfite Conversion Of Cytosine To Uracil” (U.S. applicationSer. No. 60/499,106 filed Aug. 29, 2003, and also application Ser. No.60/523,054 having the same title and filed Nov. 17, 2003), and “Methodand Materials for Bisulfite Conversion of Cytosine to Uracil (U.S.application Ser. No. 60/499,082 filed Aug. 29, 2003, and alsoapplication Ser. No. 60/523,056 (5180P2) having the same title and filedNov. 17, 2003), all of which are hereby incorporated by reference intheir entirety.

In one embodiment, the present invention contemplates labeling cytosinebases in methylated CpG dinucleotides. U.S. Pat. No. 7,285,394, herebyincorporated by reference, describes that 5-methylcytosine DNAglycosylase, in combination with art-recognized DNA repair enzymes, andin particular embodiments with DNA methyltransferase, to specificallylabel cytosine bases in methylated CpG dinucleotides in genomic DNAsequences. Such labeling occurs through enzymatic substitution of5-methylcytosine with labeled cytosine, and allows, inter alia, forselection and cloning of sequences originally containing methylated CpGdinucleotides.

EXPERIMENTAL

The following examples illustrate embodiments of the invention, butshould not be viewed as limiting the scope of the invention.

Example 1 Isolation and Photo-Release of Protein Produced in a Cell-FreeExpression System Using Incorporated PC-biotin

Cell-Free Expression and tRNA Mediated Labeling:

Glutathione-s-transferase (GST) was expressed in a cell-free reactionand co-translationally labeled using AmberGen'sPC-biotin-tRNA^(COMPLETE) (PC-biotin=photocleavable biotin) andBODIPY-FL-tRNA^(Lys) misaminoacylated tRNA reagents. AmberGen'sBODIPY-FL-tRNA^(Lys) misaminoacylated tRNA and PC-biotin reagents aredescribed in the scientific literature [Gite et al. (2003) NatBiotechnol 21, 194-197; Olejnik et al. (1995) Proceedings of theNational Academy of Science (USA) 92, 7590-7594]. Although not used inthis Example, BODIPY-FL-tRNA^(COMPLETE) is also used in later Examplesinstead of BODIPY-FL-tRNA^(Lys). “tRNA^(Lys)” refers to a purepreparation of E. coli lysine specific aminoacyl tRNA that is conjugatedto the BODIPY-FL or PC-biotin label at the ε-amine group of the aminoacid side chain. “RNA^(COMPLETE)” refers to a complete mixture of yeasttRNAs (i.e. tRNAs for all 20 amino acids) that is chemicallymisaminoacylated uniformly with a lysine conjugated to the BODIPY-FL orPC-biotin label at the ε-amino group of the amino acid side chain. Thebasic chemical aminoacylation methodology used to prepare themisaminoacylated “tRNA^(COMPLETE)” reagents is described by AmberGen inthe scientific literature [Mamaev et al. (2004) Anal Biochem 326,25-32]. In brief, these specialized misaminoacylated tRNA reagents havethe ability to co-translationally incorporate the non-native labeledamino acids that they carry into cell-free expressed proteins at variouspositions and frequencies. Expression reactions were performed using atranscription/translation coupled rabbit reticulocyte lysate system(TNT® T7 Quick for PCR DNA; Promega, Madison, Wis.) with the followingmodifications to the manufacturer's instructions: Plasmid DNA was usedat a final concentration of approximately 25 ng/μL. Expression plasmidsused were either the pETBlue-2 (EMD Biosciences, Inc., San Diego,Calif.) containing a C-terminal polyhistidine and HSV epitope tag or thepIVEX-WG (Roche Applied Science, Indianapolis, Ind.) containing only aC-terminal polyhistidine tag. Gene cloning (open reading frames) intothe expression plasmids was performed according to the manufacturer'sinstructions and plasmid amplification/isolation achieved using standardmolecular biology practices. For plasmid expression in the cell-freereaction, a complete amino acid mixture was added to a finalconcentration of 50 μM each. The final concentrationPC-biotin-tRNA^(COMPLETE) and BODIPY-FL-tRNA^(Lys) was 1 μM and 0.6 μMrespectively. Total expression reaction volume was 200 μL per sample.The reaction was carried out for 30 min at 30° C. and stopped bychilling on an ice bath and the addition of equal volume of TranslationDilution Buffer (TDB) [2×PBS pH 7.5, 2 mM DTT, 0.2% (w/v) BSA and 0.4%(v/v) of a mammalian protease inhibitor cocktail (cocktail in DMSO,Sigma-Aldrich, St. Louis, Mo.)] for a final 400 μL volume per sample(PBS=50 mM sodium phosphate pH 7.5 and 100 mM NaCl). The stoppedtranslations were equilibrated at +4° C. for 15 min and clarified byspinning 1 min 13,000 rpm in a micro-centrifuge prior to furtherprocessing. The fluid supernatant containing the soluble material waskept and used in the subsequent steps and the insoluble pellet wasdiscarded.

Isolation of Labeled Nascent Proteins:

PC-biotin labeled nascent GST was captured and isolated on 10 μL packedbead volume of NeutrAvidin agarose beads having an approximate biotinbinding capacity of 800 pmoles (Pierce Biotechnology, Inc., Rockford,Ill.). The isolation procedure was performed in batch mode using amicro-centrifuge and polypropylene tubes to manipulate the affinitymatrix and exchange the buffers. All steps were performed at +4° C. oron an ice water bath and all reagents and samples were also kept underthese conditions during the procedure. After capture on the NeutrAvidinbeads for 1 hr, beads were washed by mixing 2× briefly (briefly=5 secvortex mix) and 2× for 5 min in 45 bead volumes per wash. The bufferused for washing the beads was PBS pH 7.5, 1 mM DTT and 0.1% (w/v) BSA.Prior to photo-release of the captured and isolated GST, the washedpellet of 10 μL of NeutrAvidin agarose beads was suspended in a finalvolume of 400 μL thereby keeping the volume equal to the volume ofstarting material (i.e. volume just prior to addition of sample toNeutrAvidin agarose beads).

Photo-Release:

Photo-release of the captured GST was achieved via illumination of theNeutrAvidin bead suspension, with mixing, for 5 min with near-UV light(365 nm peak UV lamp, Blak-Ray Lamp, Model XX-15, UVP, Upland, Calif.)at a 5 cm distance. Importantly, light illumination was performeddirectly in uncovered/uncapped polypropylene micro-centrifuge tubes,such that there was no solid barrier between the bead suspension and thelight source. The power output under these conditions was 2.6 mW/cm² at360 nm, 1.0 mW/cm² at 310 nm and 0.16 mW/cm² at 250 nm. Fractions (i.e.fluid supernatant with no beads) were collected at each step of theisolation and photo-release procedures and GST content was analyzed bystandard SDS-PAGE and imaging of the fluorescent BODIPY labels using aFluorimager SI laser-based gel scanner (Molecular Dynamics/AmershamBiosciences Corp., Piscataway, N.J.).

Results:

Results are shown in FIG. 1A. Lane 1 is the initial unbound fractioncorresponding to nascent GST not binding the NeutrAvidin beads (washfactions were also collected and analyzed but contained negligiblequantities). Lane 2 is the negative control elution in the absence ofthe proper light. Lane 3 is the photo-released fraction followingillumination with the proper light. Lane 4 is the fraction remainingbound to the beads that was subsequently released by denaturation of theNeutrAvidin (asterisk indicates 2× more loading to gel relative to otherlanes). Quantification of the gel shows 81% of the total GST does notbind the NeutrAvidin beads for a calculated binding of 19%. 68% of thebound GST is photo-released with light for a 13% overall recovery. Forthe negative control, in the absence of the proper light, 3% of thebead-bound GST “leaks” from the affinity matrix.

Example 2 Isolation and Photo-Release of Protein Produced in a Cell-FreeExpression System Using Photocleavable Antibodies Preparation of aPhotocleavable Antibody Affinity Matrix:

A “photocleavable” antibody (PC-antibody) is defined, in all Examplesprovided, as an antibody conjugated to a photocleavable chemical linker,in this case photocleavable biotin (PC-biotin), that mediates attachmentof the antibody to a solid affinity matrix [in this case (strept)avidincoated beads] in a photo-reversible fashion. With proper lighttreatment, the antibody is photo-released from the solid affinitymatrix, with the antibody intact and still bound to any antigen that wasbound prior to photo-release.

400 μg of mouse monoclonal anti-HSV tag antibody (EMD Biosciences, Inc.,San Diego, Calif.) at 1 μg/μL was dialyzed extensively against 200 mMsodium bicarbonate (no pH adjustment) and 200 mM NaCl. The resultantrecovered antibody (˜200 μg at 0.3-0.4 μg/μL) was labeled using 20 molarequivalents of AmberGen's PC-biotin-NHS reagent (added from 5 mM stockin DMF) for 1 hr with mixing. The reaction was quenched for 15 min byadding one-fifth volume of a 1M glycine stock. Without additionalpurification, the resultant antibody conjugate solution is mixed 1:1with 0.1% BSA (w/v) in TBS [TBS=50 mM Tris(2-amino-2-(hydroxymethyl)-1,3-propanediol) pH 7.5 and 200 mM NaCl] andcaptured on NeutrAvidin agarose beads (Pierce Biotechnology, Inc.,Rockford, Ill.) at a ratio of 0.25 μg of antibody conjugate per μL ofpacked beads. Capture is allowed to proceed for 30 min with mixing.Beads are washed 4×5 min with 10 bead volumes each wash using 0.1% BSA(w/v) in TBS and resuspended to a 50% slurry (v/v) in the same buffer.Sodium azide is added as a preservative to 1.5 mM and the beads storedprotected from light at +4° C. Cell-Free Expression and TRNA MediatedLabeling.

Glutathione-s-transferase (GST) containing an HSV epitope tag on the C—,terminus was expressed in a cell-free reaction as described earlier inExample 1 except that only AmberGen's BODIPY-FL-tRNA^(COMPLETE) was usedat 1 μM for labeling.

Isolation of Labeled Nascent Proteins and Photo-Release:

Isolation and photo-release of GST was performed as described earlier inExample 1 except that the anti-HSV photocleavable antibody affinitymatrix was substituted for the NeutrAvidin beads in Example 1.

Results:

Results are shown in FIG. 1B. Lane 1 is the initial unbound fractioncorresponding to nascent GST not binding the photocleavable antibodybeads (wash factions were also collected and analyzed but containednegligible quantities). Lane 2 is the negative control elution in theabsence of the proper light. Lane 3 is the photo-released fractionfollowing illumination with the proper light. Lane 4 is the fractionremaining bound to the beads that was subsequently released bydenaturation of the antibody (asterisk indicates 2× more loading to gelrelative to other lanes). Quantification of the gel shows 25% of thetotal GST does not bind the photocleavable antibody beads for acalculated binding of 75%. 78% of the bound GST is photo-released withlight for a 58% overall recovery. For the negative control, in theabsence of the proper light, 3% of the bead-bound GST “leaks” from theaffinity matrix.

Example 3 Purity of Proteins Isolated by Incorporated PC-Biotin andPhoto-Released

Cell-Free Expression and tRNA Mediated Labeling:

Glutathione-s-transferase (GST) was expressed in a cell-free reaction asdescribed earlier in Example 1 except that the Translation DilutionBuffer (TDB) was modified as follows: i) DTT was not used, ii) 4 mMcycloheximide was included to ensure the expression reaction iscompletely stopped and iii) 0.02% (w/v) Triton X-100 detergent was usedas a carrier instead of BSA to avoid interference with purity analysis.

Isolation of Labeled Nascent Proteins and Photo-Release.

Isolation and photo-release of GST was performed as described earlier inExample 1 except that 0.01% (w/v) Triton X-100 detergent was used as acarrier in all buffers instead of BSA to avoid interference with purityanalysis. Additionally, to ensure detection of all possiblecontaminants, the volume of buffer used during photo-release was reducedsuch that the isolated GST was concentrated by a factor of approximately5.

Electrophoresis Based Analysis of Purity:

20 μL of purified and concentrated photo-released GST was separatedusing standard SDS-PAGE (8-16% gradient gel for comprehensive coverage)(FIG. 2). The electrophoretic gel was scanned for the selectivefluorescent labeling of nascent GST (FIG. 2A) as described in Example 1.The gel was subsequently stained for total protein using a highsensitivity silver stain method according to published reports [Sinha etal. (2001) Proteomics 1, 835-840] as shown in FIG. 2B.

Results:

Results are shown in FIG. 2. Lane 1 is plain SDS-PAGE gel loading bufferas a negative control. Lane 2 is the plain buffer used in the isolationas a negative control. Lane 3 is a negative control corresponding to thephoto-released fraction derived from a cell-free expression reactionwhere only the added DNA (GST gene in plasmid) was omitted. Lane 4 isthe photo-released fraction derived from a cell-free expression reactionwhere the GST DNA was included. The data show a GST band present only inLane 4 as expected. The asterisk denotes an unknown global contaminationoriginating either in the electrophoretic gel itself or the SDS-PAGEloading buffer but not attributable to the cell-free expressed samplesor isolation process. Disregarding the global contaminant, the GST bandis shown to be highly pure with only a few contaminating bands ofnegligible relative intensities (all contaminant bands >10-fold weakerthan GST band).

Example 4 Yield of Proteins Isolated by Incorporated PC-Biotin andPhoto-Released Western Blot Analysis of Absolute Yield:

Various human proteins were expressed and labeled in a rabbitreticulocyte cell-free reaction system, captured and photo-released inpure form as described in Example 1. In cases where co-migration duringSDS-PAGE of the BSA carrier used in the isolation procedure (66 kDa)with the expressed test protein was of concern, the BSA carrier wasreplaced with a β-casein carrier (˜24 kDa) to avoid this. Afterisolation and photo-release, test proteins were separated by standardSDS-PAGE and analyzed using standard Western blotting practices. Westernblotting was achieved with antibodies either to endogenous epitopes orto the HSV epitope tag present at the C-terminus of most expressedproteins. Linearity of the Western blot signals and quantification ofthe isolated test proteins was achieved by generating standard curvesfrom known quantities of purified commercial recombinant proteins (e.g.recombinant human PKA from Invitrogen Corporation, Carlsbad, Calif. andrecombinant Firefly luciferase from Promega, Madison, Wis.) or knownquantities of a recombinant protein bearing the HSV epitope tag (EMDBiosciences, Inc., San Diego, Calif.).

Results:

Results indicate yields of 522 pg (luciferase), 399 pg (human c-jun),267 pg (human p53), 132 pg (human MDM2), 383 pg (human PKA_(cα)) and 247pg (human GST A2) per every μL of cell-free expression reaction for anoverall average yield of 325±137 pg/μL across all tested proteins.

Example 5 Contact Photo-Transfer of Cell-Free Expressed Proteins fromBeads to Solid Surfaces Using Incorporated PC-Biotin: UV LightDependence

Cell-Free Expression and tRNA Mediated Labeling:

The human p53 oncoprotein (tumor antigen) was expressed and labeled in arabbit reticulocyte cell-free reaction system as described in Example 1with the following exceptions: PC-biotin-tRNA^(COMPLETE) was used at 2μM instead of 1 μM. The BODIPY-FL-tRNA^(Lys) was not used. Theexpression reaction carried out for 1 hr instead of 30 min. Thecomposition of the Translation Dilution Buffer (TDB) was 2×TBS, pH 7.5,0.2% (w/v) Triton X-100 and 20 mM EDTA.

Isolation of Labeled Nascent Proteins by Incorporated PC-Biotin andContact Photo-Transfer:

PC-biotin labeled nascent p53 was captured and isolated on 50 μL packedbead volume of NeutrAvidin agarose beads (Pierce Biotechnology, Inc.,Rockford, Ill.). All steps were performed at +4° C. The isolationprocedure was performed in batch mode using a micro-centrifuge andpolypropylene tubes to manipulate the affinity matrix and exchange thebuffers. After capture on the NeutrAvidin beads for 30 min, beads werewashed by mixing 3× for 5 min each in TBS pH 7.5, 0.1% (w/v) TritonX-100, 10 mM EDTA and then washed 3× briefly (briefly=5 sec vortex mix)in PBS all at 20 bead volumes per wash. Lastly, the beads were washed 2×briefly (briefly=5 sec vortex mix) with 100 bead volumes each of 40%glycerol in PBS and resuspended to a 10% bead suspension (v/v) in thesame glycerol/PBS buffer.

For contact photo-transfer, the beads were resuspended by mixing and 1μL of the bead suspension was manually pipetted onto the surface of anamine-reactive aldehyde activated glass microarray substrate (i.e.activated glass slide) (SuperAldehyde substrates, TeleChemInternational, Inc. ArrayIt™ Division, Sunnyvale, Calif.). Thesubstrates were then illuminated, without agitation, for 5 min withnear-UV light (365 nm peak UV lamp, Blak-Ray Lamp, Model XX-15, UVP,Upland, Calif.) at a 5 cm distance to photo-release and transfer the p53protein. The power output of the lamp under these conditions was 2.6mW/cm² at 360 nm, 1.0 mW/cm² at 310 nm and 0.16 mW/cm² at 250 nm. As anegative control, replicate samples on the same substrate were protectedfrom the incident UV light. After light treatment, the glass substrateswere incubated for 30 min at 37° C. in a sealed and humidified chamberto fully ensure photo-released proteins react with the activated solidsurface. The beads and any unbound protein were then washed away and thesubstrates simultaneously blocked in TBS, pH 7.5, 0.05% (w/v) Tween-20(TBS-T) plus 5% BSA (w/v) for 15 min at 37° C. Importantly,phase-contrast light microscopy reveals that the easily visible ˜100 μmagarose beads do not remain bound to any of the solid surfaces tested(see later examples for different surfaces).

Detection of Photo-Transferred Protein:

Contact photo-transferred p53 was detected on the glass substrate byprobing with a mouse monoclonal antibody against the HSV epitope tag(EMD Biosciences, Inc., San Diego, Calif.) present at the C-terminal ofthe protein. This was followed by probing with a fluorescent AlexaFluor® 488 conjugated secondary antibody (Invitrogen Corporation,Carlsbad, Calif.). Unbound antibody was washed away and the substrateswere imaged using a FluorImager SI laser-based scanner (MolecularDynamics/Amersham Biosciences Corp., Piscataway, N.J.).

Results:

Results are shown in FIG. 3 and quantification of the image shows that94% of the total signal is dependent on illumination with the properlight while only 6% of the p53 protein is transferred without light.

Example 6 Incorporated PC-Biotin: Contact Photo-Transfer Versus Releasefrom Beads into Solution Followed by Mechanical Protein Array Printing

Cell-Free Expression and tRNA Mediated Labeling:

Various human proteins were expressed and labeled in a rabbitreticulocyte cell-free reaction system as described in Example 1 withthe following exceptions: PC-biotin-tRNA^(COMPLETE) was used at 2 μMinstead of 1 ZM. The BODIPY-FL-tRNA^(Lys) was not used. The expressionreaction carried out for 1 hr instead of 30 min. The composition of theTranslation Dilution Buffer (TDB) was 2×TBS, pH 7.5, 0.2% (w/v) TritonX-100 and 20 mM EDTA. Furthermore, the cell-free expression reactionsize for each protein was varied to normalize for the differences inexpression yield.

Isolation of Labeled Nascent Proteins by) Incorporated PC-Biotin andContact Photo-Transfer:

Performed as described in Example 5. Additionally, as a comparison tocontact photo-transfer, an aliquot of the bead suspension (at the samebead to fluid ratio) containing the captured proteins was illuminatedoff-line (i.e. separately prior to application to surface) in lowprotein binding 1.5 mL polypropylene micro-centrifuge tubes (MaxymumRecovery Tubes; Axygen Scientific, Inc., Union City, Calif.) withmixing. Light illumination was otherwise performed under the sameconditions described in Example 5. Importantly, light illumination wasperformed in uncovered/uncapped tubes, such that there was no solidbarrier between the bead suspension and the light source. Note that noprotein carriers were used during photo-release (e.g. BSA) in order tofacilitate direct covalent immobilization of the isolated protein on theamine-reactive activated microarray substrate. After photo-release, thebeads were spun down in a micro-centrifuge and only the fluidsupernatant was pipetted (“printed”) onto the microarray substrate. Allsubsequent procedures were the same as for the contact photo-transferdescribed in Example 5.

Detection of Photo-Transferred Protein:

Detection of the common C-terminal HSV epitope tag was performed asdescribed in Example 5.

Results:

Results are shown in FIG. 4. The 5 contact photo-transferred proteinswere CK=casein kinase II; MDM=ubiquitin-protein ligase E3 MDM2;p53=cellular tumor antigen p53; PKA=protein kinase A catalytic subunitalpha; Tub=alpha-tubulin. Averaged over all 5 proteins, the contactphoto-transfer method achieves 9±3 fold more protein transferred to andimmobilized on the microarray substrate as compared to the method ofphoto-release into solution then immobilization. Contact photo-transferalso avoids the need for proteinaceous carriers (additives), normallyused in solution to prevent losses of the target protein vianon-specific adsorption (e.g. to the walls of the storage vial/tube).The lack of a need for proteinaceous carriers facilitates efficientimmobilization on protein binding surfaces, such the aldehyde activatedglass slides in this Example (or other surfaces such as epoxy activatedor PVDF, polystyrene or nitrocellulose surfaces or membranes/films),without competition for binding from the carrier. It also eliminates theneed for chemical carriers like detergents which my harm protein foldingand function. Improved protein transfer/immobilization can be attributedto i) since the protein is directly transferred from the beads to thesurface, no non-specific loss (adsorption) of the protein occurs on thewalls of a storage vial/tube, in the absence of a carrier; ii) thetarget protein is maintained in high concentration on the bead whichrests on the microarray surface, upon photo-release the protein is at ahigh local concentration near the binding surface and thus moreefficiently captured/immobilized. Experiments involving contactphoto-transfer from individually resolved beads shown later in FIGS. 12,13 and 14 (Examples 14, 15 and 16) further support that the protein islargely captured on the surface prior to diffusion into the fluidmedium. In contrast, pre-photo-release into solution pre-dilutes theprotein prior to application to the microarray surface; iii) betterlight delivery as the beads form a monolayer on the microarraysubstrate.

Example 7 Contact Photo-Transfer to Activated Microarray Surfaces UsingIncorporated PC-Biotin: Detection of a tRNA Mediated Direct FluorescenceLabel Cell-Free Expression and TRNA Mediated Labeling:

Human calmodulin and alpha-tubulin were expressed in a rabbitreticulocyte cell-free reaction and co-translationally labeled with bothBODIPY-FL and PC-biotin as in Example 1 with the following exceptions:BODIPY-FL-tRNA^(COMPLETE) was used for fluorescence labeling instead ofBODIPY-FL-tRNA^(Lys). As a negative control, an expression reaction wasperformed lacking only the added DNA for the gene of interest (Minus DNAblank). The Translation Dilution Buffer (TDB) used to stop the reactionand prepare the sample contained no BSA or any other protein carriers.

Isolation of Labeled Nascent Proteins:

The isolation procedure only (see later for contact photo-transfer) wasperformed as in Example 1 with the following exceptions: The buffersused in the procedure contained no BSA or other protein carriers at anystep. Capture on the NeutrAvidin beads was for 30 min. After washing theunbound material from the NeutrAvidin beads as described in Example 1the beads were further washed 3× briefly (briefly=5 sec vortex mix) with45 bead volumes each of plain PBS and 1×5 min with 45 bead volumes of40% glycerol in PBS. The washed bead pellet was then suspended withequal volume of 40% glycerol in PBS to yield a 50% bead slurry (v/v).

Contact Photo-Transfer:

For contact photo-transfer, the beads were resuspended by mixing and 1μL of the bead suspension was manually pipetted onto the surface of areactive epoxy activated glass microarray substrate (i.e. activatedglass slide) (SuperEpoxy substrates, TeleChem International, Inc.ArrayIt™ Division, Sunnyvale, Calif.). Note that 1 μL of bead suspensiondeposited on the substrate (corresponding to one spot in FIG. 5)contained roughly 400 agarose beads prior to removal/washing. Thesubstrates were then illuminated, without agitation, for 5 min withnear-UV light (365 nm peak UV lamp, Blak-Ray Lamp, Model XX-15, UVP,Upland, Calif.) at a 5 cm distance to photo-release and transfer thetarget proteins. The power output of the lamp under these conditions was2.6 mW/cm² at 360 nm, 1.0 mW/cm² at 310 nm and 0.16 mW/cm² at 250 nm.After light treatment, the glass substrates were incubated for 30 min at37° C. in a sealed and humidified chamber to fully ensure photo-releasedproteins react with the activated solid surface. The beads and anyunbound protein are then washed away from the microarray substratesurface, in a tray, with several rounds of excess buffer (e.g. 20 mL persubstrate of TBS or PBS with or without 0.05% w/v Tween-20 detergent).Phase contrast light microscopy reveals that the easily visible 100micron NeutrAvidin agarose beads were completely washed/removed from theglass substrates. In fact, when 1 μL of a 50% (v/v) bead suspension isapplied per spot to the glass substrates, the bead monolayer is evenplainly visible by eye prior to washing/removal without the need for amicroscope; and the monolayer is clearly observed to be gone immediatelyafter submersion even in the first wash. The glass substrates werefurther rinsed in excess purified water to remove salts prior to dryingand imaging.

Detection of Photo-Transferred Protein:

Detection of the directly incorporated tRNA mediated BODIPY-FLfluorescence labeling was achieved by imaging the dry microarraysubstrates on an ArrayWoRx^(e) BioChip fluorescence reader (AppliedPrecision, LLC, Issaquah, Wash.).

Results:

Results are shown in FIG. 5. −DNA=minus DNA blank derived fromexpression reaction lacking only the added DNA for gene of interest (allother processing steps otherwise performed same as with DNA containingexpressed protein samples); Calm calmodulin; Tub=alpha-tubulin;*=decreased sample loading to roughly normalize signal to calmodulin.The results show that the directly incorporated BODIPY-FL fluorescencelabel is easily detectible following contact photo-transfer compared tothe minus DNA blank. Signal to noise ratios exceed 6:1 in all cases andreach 29:1 in the case of tubulin (full loading). Note that calmodulinbinds relatively poorly to the glass substrate due to it's highly acidicnature (pI=3; low lysine content; epoxy activated surfaces primarilyreact with primary amines).

Example 8 Contact Photo-Transfer to 3-Dimensional Matrix CoatedMicroarray Surfaces Using Incorporated PC-Biotin: Detection of a tRNAMediated Direct Fluorescence Label

Cell-Free Expression and tRNA Mediated Labeling:

Human calmodulin and alpha-tubulin were expressed in a rabbitreticulocyte cell-free reaction and co-translationally labeled with bothBODIPY-FL and PC-biotin as in Example 1 with the following exceptions:BODIPY-FL-tRNA^(COMPLETE) was used for fluorescence labeling instead ofBODIPY-FL-tRNA^(Lys). As a negative control, an expression reaction wasperformed lacking only the added DNA for the gene of interest (Minus DNAblank). The Translation Dilution Buffer (TDB) used to stop the reactionand prepare the sample contained no BSA or any other protein carriers.

Isolation of Labeled Nascent Proteins:

The isolation procedure only (see later for contact photo-transfer) wasperformed as in Example 1 with the following exceptions: The buffersused in the procedure contained no BSA or other protein carriers at anystep. Capture on the NeutrAvidin beads was for 30 min. After washing theunbound material from the NeutrAvidin beads as described in Example 1the beads were further washed 3× briefly (briefly=5 sec vortex mix) with45 bead volumes each of plain PBS and 1×5 min with 45 bead volumes of40% glycerol in PBS. The washed bead pellet was then suspended withequal volume of 40% glycerol in PBS to yield a 50% bead slurry (v/v).

Contact Photo-Transfer:

Performed as in Example 7 with the following exceptions: Proteins werecontact-photo transferred onto 3-dimensional polyacrylamide basedHydroGel coated microarray substrates (PerkinElmer Life and AnalyticalSciences, Inc., Boston, Mass.). Prior to contact photo-transfer, theHydroGel slides were re-hydrated according to the manufacturersinstructions. Following contact photo-transfer, the proteins wereallowed to bind to the HydroGel matrix for overnight at +4° C. prior towashing away the beads and unbound material.

Detection of Photo-Transferred Protein:

Detection of the directly incorporated tRNA mediated BODIPY-FLfluorescence labeling was achieved as described in Example 7.

Results:

Results are shown in FIG. 6. −DNA=minus DNA blank derived fromexpression reaction lacking only the added DNA for gene of interest (allother processing steps otherwise performed same as with DNA containingexpressed protein samples); Calm=calmodulin; Tub=alpha-tubulin. Theresults show that the directly incorporated BODIPY-FL fluorescence labelis easily detectable following contact photo-transfer compared to theminus DNA blank. Signal to noise ratios are 4:1 in both cases.Compatibility with matrix coated surfaces such as the HydroGelsubstrates is important as they may more effectively maintain functionof the immobilized proteins versus the essentially flat solid glasssubstrates for example. Better maintenance of protein function isexpected, for example, with surfaces that are hydrophilic, do notchemically react with the protein, and/or maintain the protein in ahydrated state.

Example 9 Contact Photo-Transfer From Magnetic Beads to Antibody CoatedMicroarray Surfaces Using Incorporated PC-Biotin: Detection of a tRNAMediated Direct Fluorescence Label

Cell-Free Expression and tRNA Mediated Labeling:

Human GST was expressed in a rabbit reticulocyte cell-free reaction andco-translationally labeled with both BODIPY-FL and PC-biotin as inExample 1 with the following exceptions: BODIPY-FL-tRNA^(COMPLETE) wasused for fluorescence labeling instead of BODIPY-FL-tRNA^(Lys). As anegative control, an expression reaction was performed lacking only theadded DNA for the gene of interest (Minus DNA blank). The TranslationDilution Buffer (TDB) used to stop the reaction and prepare the samplealso contained 0.02% Triton X-100 detergent (w/v) in addition to the0.2% BSA (w/v) as carriers to prevent non-specific adhesion oraggregation of the 1 micron magnetic beads. TDB was also supplementedwith 4 mM cycloheximide.

Isolation of Labeled Nascent Proteins:

The isolation procedure only (see later for contact photo-transfer) wasperformed as in Example 1 with the following exceptions: The buffersused in the procedure contained no BSA or other protein carriers at anystep. Capture on the NeutrAvidin beads was for 30 min. After washing theunbound material from the NeutrAvidin beads as described in Example 1the beads were further washed 3× briefly (briefly=5 sec vortex mix) with45 bead volumes each of plain PBS and 1×5 min with 45 bead volumes of40% glycerol in PBS. The washed bead pellet was then suspended withequal volume of 40% glycerol in PBS to yield a 50% bead slurry (v/v).

Furthermore, all Examples prior to this used NeutrAvidin conjugatedcross-linked agarose beads (˜100 micron) as the affinity matrix forcapture of the PC-biotin labeled expressed proteins. However, magneticbeads are desirable due to their ease of manipulation with magneticdevices and are readily available in various relatively small anduniform sizes. In this example, proteins were captured/isolated onstreptavidin conjugated 1 micron diameter magnetic beads (Dynabeads®MyOne™ Streptavidin; Dynal Biotech LLC, Brown Deer, Wis.). 114 μg ofbeads was used for each sample which corresponds to roughly 1×10⁸ beadswith a biotin binding capacity of approximately 400 pmoles. For allprocessing steps, beads were separated from the fluid in thepolypropylene micro-centrifuge tubes using the appropriate manufacturersupplied magnetic device. In contrast to Examples 1 and 7 involvingagarose beads, the buffer used during capture on the streptavidinmagnetic beads contained both 0.1% BSA (w/v) and 0.01% Triton X-100detergent (w/v) as carriers to prevent non-specific adhesion oraggregation of the beads. Also in contrast to Examples 1 and 7,following capture of the target protein on the beads, the full washingregimen was as follows: 2× briefly (briefly 5 sec vortex mix) and 2×5min in 0.5 mL per sample of 1 mM DTT, 0.1% w/v BSA and 0.01% w/v TritonX-100 in PBS then 1× briefly (briefly=5 sec vortex mix) in 0.5 mL persample of 0.1% BSA w/v in PBS and 1× briefly (briefly=5 sec vortex mix)in 1 mL per sample of plain PBS. Lastly, each washed bead pellet wassuspended in 45 μL (˜2.5 μg/μL bead concentration) of 50% glycerol and1% BSA w/v in PBS.

Preparation of Anti-HSV Monoclonal Antibody Coated MicroarraySubstrates:

The commercially available mouse monoclonal anti-HSV tag antibody (EMDBiosciences, Inc., San Diego, Calif.) at 1 μg/μL was diluted ⅛ in PBSand 64 μL was applied to reactive epoxy activated glass microarraysubstrates (i.e. activated glass slide) (SuperEpoxy substrates,TeleChein International, Inc. ArrayIt™ Division, Sunnyvale, Calif.). Thesolution was spread evenly over the substrate surface by overlaying a22×60 mm cover glass. Binding to the surface was allowed to occur for 30min at 37° C. in a humidified chamber without agitation. Slides werethen washed 4×2 min with excess (>20 mL) TBS-T and blocked in TBS-Tsupplemented freshly with 0.1M glycine. Slides were rinsed 4× briefly (5sec) in purified water and dried.

Contact Photo-Transfer:

Performed as in Example 7 except that the 1 micron streptavidin magneticbeads were used here and transfer and immobilization was onto theanti-HSV monoclonal antibody coated microarray substrates. Note that 1μL of bead suspension deposited onto the substrate (corresponding to onespot in FIG. 7) contained roughly 2×10⁶ beads prior to removal/washing.Phase contrast light microscopy reveals that the 1 micron magneticbeads, also visible under the microscope, were completely washed/removedfrom the glass substrates. However, omission of the BSA carrier from thecontact photo-transfer buffer results in non-specific adhesion of the 1micron streptavidin magnetic beads to the substrate surface, unlike withthe 100 micron NeutrAvidin agarose beads.

Detection of Photo-Transferred Protein:

Performed as in Example 7.

Results:

Results are shown in FIG. 7. −DNA=minus DNA blank derived fromexpression reaction lacking only the added DNA for gene of interest (allother processing steps otherwise performed same as with DNA containingexpressed protein samples); GST=glutathione-s-transferase. The resultsshow that the directly incorporated BODIPY-FL fluorescence label iseasily detectible following contact photo-transfer compared to the minusDNA blank with a signal to noise ratio of 9:1. This example importantlydemonstrates 2 achievements, i) contact photo-transfer from magneticbeads which are desirable due to their readily available small anduniform sizes and their ease of manipulation for automated assays forexample; ii) contact photo-transfer onto antibody coated microarraysubstrates rather than chemically reactive substrates (e.g. epoxy).

Example 10 Photo-Transfer to Polystyrene 96-Well Microtiter Plates UsingIncorporated PC-Biotin: Detection by Antibody

Cell-Free Expression and tRNA Mediated Labeling

Human p53 oncoprotein (tumor antigen) and alpha-tubulin proteins wereexpressed and labeled in a rabbit reticulocyte cell-free reaction systemas described in Example 1 with the following exceptions:PC-biotin-tRNA^(COMPLETE) was used at 3 μM instead of 1 μM. TheBODIPY-FL-tRNA^(Lys) was not used. 100 μL of total expression reactionwas used instead of 200 μL. The expression reaction carried out for 1 hrinstead of 30 min. The composition of the Translation Dilution Buffer(TDB) was 2×TBS, pH 7.5, 0.2% (w/v) Triton X-100 and 20 mM EDTA.

Isolation of Labeled Nascent Proteins:

PC-biotin labeled nascent proteins were captured and isolated on 50 μLpacked bead volume of NeutrAvidin agarose beads (Pierce Biotechnology,Inc., Rockford, Ill.). The isolation procedure was performed in batchmode using a micro-centrifuge and polypropylene tubes to manipulate theaffinity matrix and exchange the buffers. After capture on theNeutrAvidin beads for 1 hr, beads were washed by mixing 3× briefly(briefly=5 sec vortex mix) in TBS pH 7.5, 0.1% (w/v) Triton X-100 and 10mM EDTA at 20 bead volumes per wash. Beads were then washed 3× briefly(briefly=5 sec vortex mix) in 50 mM sodium carbonate, pH 9.5 and 50 mMNaCl at 40 bead volumes per wash and lastly prepared to a 5% beadsuspension (v/v) in the same buffer.

Photo-Transfer to Wells of a Microtiter Plate:

100 μL/well of the 5% bead suspension, corresponding to each capturedtarget protein, was loaded into opaque white polystyrene 96-wellmicrotiter plates (Microlite 2+; Thermo Labsystems, Franklin, Mass.) forphoto-release and subsequent protein adsorption (transfer) to thepolystyrene well surface. For photo-release, the plate was illuminatedfrom the top for 5 min with near-UV light (365 nm peak UV lamp, Blak-RayLamp, Model XX-15, UVP, Upland, Calif.) at a 5 cm distance while mixingon an orbital plate shaker. The power output of the lamp under theseconditions was 2.6 mW/cm² at 360 nm, 1.0 mW/cm² at 310 nm and 0.16mW/cm² at 250 nm. After light treatment, mixing was continued for 1 hrto allow the photo-released proteins to bind the well surface.

Detection of Photo-Transferred Protein:

For detection purposes, the commercially available mouse monoclonalanti-HSV tag antibody (EMD Biosciences, Inc., San Diego, Calif.) wasconjugated to an alkaline phosphatase enzyme reporter using acommercially available maleimide activated alkaline phosphatase reagent(Pierce Biotechnology, Inc., Rockford, Ill.) essentially according tothe manufacturer's instructions.

Following photo-transfer of the target proteins to the microtiter platewells, the bead suspension was removed and the wells washed 4× briefly(5 sec) in 300 μL/well of TBS-T. Wells were then blocked for 30 min in5% BSA (w/v) in TBS-T. Detection was achieved by adding the anti-HSValkaline phosphatase conjugate at 0.1 ng/μL in 5% BSA (w/v) in TBS-T for30 min. Plates were washed again and signal was generated using acommercially available chemiluminescence alkaline phosphatase substrate(Roche Applied Science, Indianapolis, Ind.) according to themanufacturer's instructions. Signal was read in a LumiCount luminescenceplate reader (Packard/PerkinElmer Life and Analytical Sciences, Inc.,Boston, Mass.).

Results:

Results are shown in FIG. 8. −DNA=minus DNA blank derived fromexpression reaction lacking only the added DNA for gene of interest (allother processing steps otherwise performed same as with DNA containingexpressed protein samples); Tubulin=alpha-tubulin; p53=cellular tumorantigen p53; RLU=raw relative luminescence units. The results show cleardetection of the photo-transferred proteins by way of the commonC-tcrminal HSV epitope tag with signal to noise ratios of 140:1 and609:1 for alpha-tubulin and p53 respectively compared to the minus DNAnegative control sample.

Example 11 Photo-Transfer to Antibody Coated 96-Well Microtiter PlatesUsing Incorporated PC-Biotin: Detection by Antibody in Sandwich ELSIAFormat

Cell-Free Expression and tRNA Mediated Labeling:

Human p53 oncoprotein (tumor antigen) was expressed and labeled in arabbit reticulocyte cell-free reaction system as described in Example 1with the following exceptions: PC-biotin-tRNA^(COMPLETE) was used at 1.5μM instead of 1 μM. The BODIPY-FL-tRNA^(Lys) was used, for qualitycontrol purposes only, at 1.5 μM instead of 0.6 μM. 100 μL of totalexpression reaction was used instead of 200 μL. The expression reactioncarried out for 1 hr instead of 30 min. The composition of theTranslation Dilution Buffer (TDB) was 2×TBS, pH 7.5, 0.2% (w/v) TritonX-100 and 20 mM EDTA.

Isolation of Labeled Nascent Proteins:

PC-biotin labeled nascent p53 was captured and isolated on 10 μL packedbead volume of NeutrAvidin agarose beads (Pierce Biotechnology, Inc.,Rockford, Ill.). The isolation procedure was performed in batch modeusing a micro-centrifuge and polypropylene tubes to manipulate theaffinity matrix and exchange the buffers. After capture on theNeutrAvidin beads for 30 min, beads were washed by mixing 3×5 min eachin TBS pH 7.5, 0.1% (w/v) Triton X-100 and 10 mM EDTA at 45 bead volumesper wash. Beads were then washed 3× briefly (briefly=5 sec vortex mix)with 40% glycerol in PBS with 45 bead volumes per wash. For qualitycontrol purposes at this point, the washed bead pellets were imaged inthe tube using the FluorImager SI laser-based fluorescence scanner(Molecular Dynamics/Amersham Biosciences Corp., Piscataway, N.J.) andthe signal from the p53 sample compared to the minus DNA negativecontrol to confirm the expression and isolation was successful. The beadpellet was then further washed 3× briefly (briefly=5 sec vortex mix) inTBS-T at 45 bead volumes each and the beads ultimately prepared to anapproximate 5% suspension (v/v) in the same buffer.

Photo-Transfer to Wells of an Antibody Coated Microtiter Plate:

The commercially available mouse monoclonal anti-HSV tag antibody (EMDBiosciences, Inc., San Diego, Calif.) was adsorbed/coated onto the wellsof opaque white polystyrene 96-well microtiter plates (Microlite 2+;Thermo Labsystems, Franklin, Mass.) and the plates washed then blocked[5% BSA (w/v) in TBS-T] using standard ELISA procedures. 100 μL/well ofthe 5% bead suspension corresponding to the captured p53 protein wasloaded into the anti-HSV coated microtiter plate for photo-release andsubsequent protein capture via the C-terminal HSV epitope tag. Forphoto-release, the plate was illuminated from the top for 5 min withnear-UV light (365 nm peak UW lamp, Blak-Ray Lamp, Model XX-15, UVP,Upland, Calif.) at a 5 cm distance while mixing on an orbital plateshaker. The power output of the lamp under these conditions was 2.6mW/cm² at 360 nm, 1.0 mW/cm² at 310 nm and 0.16 mW/cm² at 250 nm. Afterlight treatment, mixing was continued for 30 min to allow thephoto-released protein to bind the antibody coated well surface.

Detection of Photo-Transferred Protein:

Following photo-transfer of the target protein to the antibody coatedmicrotiter plate wells, the bead suspension was removed and the wellswashed 4× briefly (5 sec) in 300 μL/well of TBS-T. Wells were thenblocked for 30 min in 5% BSA (w/v) in TBS-T. Detection was achieved in asandwich ELISA format with a well characterized monoclonal anti-p53horseradish peroxidase (HRP) conjugate (antibody clone BP53-12 customordered from Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.). Theantibody was added at 50 pg/μL in 5% BSA (w/v) in TBS-T for 30 min.Plates were washed again and signal was generated using a commerciallyavailable chemiluminescence HRP substrate (SuperSignal Femto ELISASubstrate; Pierce Biotechnology, Inc., Rockford, Ill.) according to themanufacturer's instructions. Signal was read in a LumiCount luminescenceplate reader (Packard/PerkinElmer Life and Analytical Sciences, Inc.,Boston, Mass.).

Results:

Results are shown in FIG. 9. −DNA=minus DNA blank derived fromexpression reaction lacking only the added DNA for gene of interest (allother processing steps otherwise performed same as with DNA containingexpressed protein samples); p53=cellular tumor antigen p53; RLU=rawrelative luminescence units. The results show clear detection of thephoto-transferred p53 with a signal to noise ratio of 719:1 compared tothe minus DNA negative control sample.

Example 12 Contact Photo-Transfer to Activated Microarray Surfaces UsingIncorporated PC-Biotin: Application to Advanced 2 Color Protein-ProteinInteraction Assays

Cell-Free Expression and tRNA Mediated Labeling:

Human calmodulin and alpha-tubulin were expressed in a rabbitreticulocyte cell-free reaction and co-translationally labeled with bothBODIPY-FL and PC-biotin as in Example 1 with the following exceptions:BODIPY-FL-tRNA^(COMPLETE) was used for fluorescence labeling instead ofBODIPY-FL-tRNA^(Lys). As a negative control, an expression reaction wasperformed lacking only the added DNA for the gene of interest (Minus DNAblank). The Translation Dilution Buffer (TDB) used to stop the reactionand prepare the sample contained no BSA or any other protein carriers.

Isolation of Labeled Nascent Proteins:

The isolation procedure only (see later for contact photo-transfer) wasperformed as in Example 1 with the following exceptions: The buffersused in the procedure contained no BSA or other protein carriers at anystep. Capture on the NeutrAvidin beads was for 30 min. After washing theunbound material from the NeutrAvidin beads as described in Example 1the beads were further washed 3× briefly (briefly=5 sec vortex mix) with45 bead volumes each of plain PBS and 1×5 min with 45 bead volumes of40% glycerol in PBS. The washed bead pellet was then suspended withequal volume of 40% glycerol in PBS to yield a 50% bead slurry (v/v).

Contact Photo-Transfer: Performed as in Example 7. Spotting of CrudeExpression Reaction to Microarray Surface for Comparison:

To demonstrate one advantage of the contact photo-transfer method, acomparison was made to microarray immobilized proteins that were notpre-purified by the incorporated PC-biotin and not applied to themicroarray surface via contact photo-transfer. Instead, proteins wereapplied to the microarray surface directly in the crude expressionreaction. Because the samples were applied in crude format, theimmobilization method could not be via a non-specific protein-reactivechemically activated substrate (e.g. epoxy activated glass substrates).Instead, the immobilization needed to be via a selective affinityinteraction. For this, microarray substrates coated with the anti-HSVepitope tag antibody were used to capture via the common C-terminalepitope tag present in the expressed proteins. This antibody was chosendue to its proven effectiveness in protein capture as demonstrated inseveral former and later Examples.

Microarray substrates were coated with the anti-HSV antibody asdescribed earlier in Example 9. Proteins were expressed as describedearlier in this Example except that only BODIPY-FL-tRNA^(COMPLETE) wasused for labeling at 4 μM and not the PC-biotin-tRNA^(COMPLETE). Aftercell-free protein expression, the reactions were diluted with equalvolume of 80% glycerol, 2 mM DTT, 20 mM EDTA and 2% (v/v) of a mammalianprotease inhibitor cocktail (Sigma-Aldrich, St. Louis, Mo.) in 2×PBS. 1μL of the prepared crude expression reactions was applied to theanti-HSV antibody coated microarray substrates and allowed to bind for 1hr at 37° C. in a humidified chamber.

Preparation of a Cy5 Conjugated Calcineurin Probe for FluorescenceDetection of Protein-Protein Interaction:

In order to measure the known biological binding interaction between themicroarray deposited calmodulin “bait” and calcineurin, a fluorescentlylabeled calcineurin-Cy5 conjugate/probe was prepared as follows: Acommercially available 100 Unit vial of calcineurin purified from bovinebrain (Sigma-Aldrich, St. Louis, Mo.; Catalog# C1907) corresponding toapproximately 20 μg was used for fluorescence labeling. Calcineurin wasdissolved in 50 μL of 200 mM sodium bicarbonate and 200 mM NaCl. A 2.7mM stock of Cy5—NHS monoreactive ester (Amersham Biosciences Corp.,Piscataway, N.J.) was prepared in DMSO fresh before use and enough addedto the calcineurin solution to achieve a 10-fold molar excess of theCy5—NHS ester. The labeling reaction was allowed to proceed by gentlemixing for 30 min protected from light with aluminum foil. 1/9^(th)volume freshly prepared 100 mM L-lysine monohydrochloride in 200 mMsodium bicarbonate and 200 mM NaCl was added to quench the reaction andmixed for 15 min protected from light with aluminum foil. BSA was thenadded from a 10% stock as a carrier to a final 0.05% (w/v)concentration. Unreacted/hydrolyzed labeling reagent, quenched labelingreagent, and L-lysine contaminants were removed from the labeledcalcineurin protein using a MicroSpin G-25 desalting column (AmershamBiosciences Corp., Piscataway, N.J.) according to the manufacturersinstructions. (except that the column was additionally pre-washed 1×350μL with TBS). Protein recovery was estimated at 0.16 μg/L and the probestock stored frozen long term at −70° C. Note that initially, a similarcalcineurin labeling procedure was done except using a BODIPY-FL-SSElabeling reagent (Invitrogen Corporation, Carlsbad, Calif.) instead ofthe Cy5—NHS monoreactive ester. This allowed analysis of the conjugatevia SDS-PAGE and fluorescence imaging of the gel using a Fluorimager SIargon laser-based scanner (Molecular Dynamics/Amersham BiosciencesCorp., Piscataway, N.J.) in order to verify successful conjugation andestimate protein concentration using known standards.

Probing the Proteins on the Microarray with the Calcineurin-Cy5Conjugate:

The calmodulin and tubulin proteins immobilized on the microarraysubstrates as described earlier in this Example were subsequently probedwith the calcineurin-Cy5 conjugate to test for its expected biologicalinteraction with calmodulin. To do so, microarray substrates were rinsed3× briefly (5 sec) with excess TBS directly following transfer andbinding to the substrate surface. The microarray substrates were thenblocked 10 min with an excess of 1% BSA (w/v) in TBS. Thecalcineurin-Cy5 probe stock prepared as described earlier in thisExample was diluted 1/30 in 1% BSA (w/v) and 2 mM CaCl₂ in TBS. Eachmicroarray substrate was probed with 75 μL of the dilutedcalcineurin-Cy5 solution by overlaying with a 22×60 mm glass coverslipand incubating for 45 min in a humidified chamber. Unbound probe wasthen removed by washing 3×1 min each with excess 2 mM CaCl₂ in TBS andthen 1× briefly (5 sec) with 2 mM CaCl₂ in purified water. Themicroarray substrates were then dried. Since the binding interaction iscalcium dependant, as a negative control, a separate permutation wasperformed whereby the CaCl₂ was omitted from all buffers where presentand replaced with the same concentration of EDTA (minus calciumpermutation).

Detection of Photo-Transferred Protein:

Detection of the directly incorporated tRNA mediated BODIPY-FLfluorescence labeling as well as binding of the fluorescentcalcineurin-Cy5 probe was achieved by imaging the dry microarraysubstrates on an ArrayWoRx^(e) BioChip fluorescence reader (AppliedPrecision, LLC, Issaquah, Wash.) using the appropriate standard built-infilter sets to discriminate between the 2 color fluorophores.

Results:

Results are shown in FIG. 10. −DNA=minus DNA blank derived fromexpression reaction lacking only the added DNA for gene of interest (allother processing steps otherwise performed same as with DNA containingexpressed protein samples); Calm=calmodulin; Tub=alpha-tubulin; ContactPhoto-Transfer=microarrays prepared by contact photo-transfer thenprobed; Crude Spotting=microarrays prepared by applying the crudecell-free expression reaction to anti-HSV antibody coated substrates andthen probed; 2 mM CaCl₂=the plus calcium calcineurin probingpermutation; 2 mM EDTA=the minus calcium calcineurin probingpermutation. In the case of the contact photo-transfer permutation, theresults clearly show the expected binding of the calcineurin probe onlyto calmodulin, in correlation with the known biological interaction asreported in the literature [Nakamura et al. (1992) FEBS Lett 309,103-106], and not tubulin. The direct tRNA mediated BODIPY-FL labelingconfirms that both calmodulin and tubulin are present on the arraysurface compared to the minus DNA control (in fact tubulin is moreabundant although it does not bind the probe as expected). Furthermore,as expected, the calcium dependant calcineurin interaction is abolishedin the absence of calcium and presence of the metal chelator EDTA.However, in the so-called “Crude Spotting” permutation, while the directtRNA mediated BODIPY-FL labeling confirms the presence of tubulin, thecalmodulin is essentially equal to background. This is likely explainedby the lack of a concentrating pre-purification step as with the contactphoto-transfer or inaccessibility of the HSV epitope due to proteinfolding. More importantly, both the BODIPY-FL and Cy5 fluorescenceimages show measurable signal in the minus DNA negative control,indicating non-specific binding of components from the crude expressionreaction to the microarray substrate that are not washed away.Importantly, these non-specifically bound contaminants, likely presentin excessive quantities, mediate non-specific binding of the calcineurinprobe to the spot areas in all applied samples, effectively masking anypotential specific signal from the calcineurin-calmodulin interaction.

Example 13 Contact Photo-Transfer to Microarray Surfaces UsingIncorporated PC-Biotin: Advanced Kinase Substrate Profiling Assays

Cell-Free Expression and tRNA Mediated Labeling:

Various human proteins were expressed and labeled in a rabbitreticulocyte cell-free reaction system as described in Example 1 withthe following exceptions: PC-biotin-tRNA^(COMPLETE) was used at 2 μMinstead of 1 μM. The BODIPY-FL-tRNA^(Lys) was not used (nor any othertRNA mediated fluorescence labeling). The volume of expression reactionfor each protein species was varied to approximately normalize fordifferences in expression efficiencies. The expression reaction carriedout for 1 hr instead of 30 min. The composition of the TranslationDilution Buffer (TDB) was 2×TBS, pH 7.5, 0.2% (w/v) Triton X-100 and 20mM EDTA.

Isolation of Labeled Nascent Proteins by Incorporated PC-Biotin andContact Photo-Transfer:

PC-biotin labeled nascent p53 was captured and isolated on 50 μL packedbead volume of NeutrAvidin agarose beads (Pierce Biotechnology, Inc.,Rockford, Ill.). All steps were performed at +4° C. The isolationprocedure was performed in batch mode using a micro-centrifuge andpolypropylene tubes to manipulate the affinity matrix and exchange thebuffers. After capture on the NeutrAvidin beads for 30 min, beads werewashed by mixing 3× for 5 min each in TBS pH 7.5, 0.1% (w/v) TritonX-100, 10 mM EDTA and then washed 2× briefly (briefly=5 sec vortex mix)in PBS all at 20 bead volumes per wash. Lastly, the beads were washed 1×briefly (briefly=5 sec vortex mix) with 20 bead volumes of 40% glycerolin PBS and resuspended to a 50% bead slurry (v/v) in the sameglycerol/PBS buffer.

For contact photo-transfer, the beads were resuspended by mixing and 1μL of the bead suspension was manually pipetted onto the surface of anamine-reactive aldehyde activated glass microarray substrate (i.e.activated glass slide) (SuperAldehyde substrates, TeleChemInternational, Inc. ArrayIt™ Division, Sunnyvale, Calif.). The slideswere then illuminated, without agitation, for 5 min with near-UV light(365 nm peak UV lamp, Blak-Ray Lamp, Model XX-15, UVP, Upland, Calif.)at a 5 cm distance to photo-release and transfer the p53 protein. Thepower output of the lamp under these conditions was 2.6 mW/cm² at 360nm, 1.0 mW/cm² at 310 nm and 0.16 mW/cm² at 250 nm. After lighttreatment, the glass slides were incubated for 30 min at 37° C. in asealed and humidified chamber to fully ensure photo-released proteinsreact with the activated solid surface. The beads and any unboundprotein were then washed away and the unreacted aldehyde groups on theslides simultaneously blocked for 15 min in 0.25% sodium borohydride(w/v) prepared immediately before use in PBS. Importantly,phase-contrast light microscopy reveals that the easily visible ˜100 μmagarose beads do not remain bound to any of the solid surfaces tested(see later examples for different surfaces). The slides were furtherwashed 4× briefly (5 sec) in excess PBS and again blocked for 15 min at37° C. with 0.1M glycine in TBS-T. Slides were rinsed 4× briefly (5 sec)in excess purified water and dried prior to further processing.

Kinase Treatment of Photo-Transferred Proteins on Microarray Slide:

Kinase solutions were prepared fresh immediately prior to use asfollows: ZAP-70 Tyrosine Kinase—979.5 μL of ZAP-70 Base Buffer [50 mMTris (2-amino-2-(hydroxymethyl)-1,3-propanediol), pH 7.0, 150 mM NaCland 10 mM MnCl₂] was further supplemented with 5 μL of a 1M MnCl₂ stock(Sigma-Aldrich, St. Louis, Mo.), 1 μL of a 1M DTT stock (stock stored inaliquots at −70° C.), 10 μL of a 10% Triton X-100 detergent stock and4.5 μL of a commercially available 230 ng/μL human recombinant ZAP-70tyrosine kinase stock (Invitrogen Corporation, Carlsbad, Calif.). Justprior to application to the microarray slide, 1 mL of this kinasemixture was supplemented with 53 μL of a 20 mM ATP stock (stock storedin aliquots at −70° C.).

Src pp⁶⁰ Tyrosine Kinase—972.2 μL of ZAP-70 Base Buffer [50 mM Tris(2-amino-2-(hydroxymethyl)-1,3-propanediol), pH 7.0, 150 mM NaCl and 10mM MnCl₂] was further supplemented with 5 μL of a 1M MnCl₂ stock(Sigma-Aldrich, St. Louis, Mo.), 1 μL of a 1M DTT stock (stock stored inaliquots at −70° C.), 10 μL of a 10% Triton X-100 detergent stock, 10 μLof a 1M MgCl₂ stock (Sigma-Aldrich, St. Louis, Mo.) and 1.8 μL of acommercially available 580 ng/μL human recombinant Src pp⁶⁰ tyrosinekinase stock (Invitrogen Corporation, Carlsbad, Calif.). Just prior toapplication to the microarray slide, 1 mL of this kinase mixture wassupplemented with 53 μL of a 20 mM ATP stock for a 1 mM final (stockstored in aliquots at −70° C.).

Dried microarray slides containing the photo-transferred proteins asdescribed earlier in this Example were overlaid with 1 mL of theaforementioned kinase mixtures and incubated for 30 min at 37° C. in ahumidified chamber. This was to allow the kinase to phosphorylate anypotential enzyme substrates (photo-transferred proteins) on themicroarray slide surface. The kinase reaction was stopped and any kinasesolution removed by washing the slides 4×2 min each with excess 10 mMEDTA in TBS-T. Any potentially bound kinase was stripped from the slidesby treating the slides for 30 min at 65° C. in a denaturing buffer [2%SDS (w/v) and 5 mM DTT in 50 mM Tris, pH 6.8]. The denaturing buffer wasremoved by washing the slides 4× briefly (5 sec) in excess TBS-T.

Detection of Phosphorylation:

To detect phosphorylation of any potential kinase substrates (i.e.phosphorylation of photo-transferred proteins) on the microarray slide,the slides were probed with a universal anti-phosphotyrosine antibody.The antibody used was a recombinant derivative of the well known andestablished PY20 monoclonal anti-phosphotyrosine antibody, thecommercially available so-called RC20 antibody clone which was suppliedlabeled with biotin to allow secondary detection (BD Biosciences, SanJose, Calif.). The microarray slides were first pre-blocked for 15 minat 37° C. with 5% BSA (w/v) in TBS-T. The RC20 anti-phosphotyrosinebiotin conjugated antibody was used at 0.5 ng/μL diluted with 5% BSA(w/v) in TBS-T and the slides treated for overnight at +4° C. withgentle mixing. After antibody binding, the slides are washed 4×2 mineach with excess TBS-T and secondary fluorescence detection is performedusing a streptavidin-Alexa Fluor® 488 dye conjugate (InvitrogenCorporation, Carlsbad, Calif.) at a concentration of 0.2 ng/μL dilutedin 5% BSA (w/v) in TBS-T. Secondary detection was performed for 1 hrwith gentle mixing. Slides were then washed 4×2 min each with excessTBS-T, rinsed 4× briefly (5 sec) in purified water and dried.

Phosphorylation Controls:

As a negative control, the aforementioned kinase reactions on themicroarray slides were performed except only the necessary ATP wasomitted from the kinase reaction mixture. Additionally, as a positivecontrol, commercially available phosphotyrosine conjugated to BSA(Sigma-Aldrich, St. Louis, Mo.) was also pre-spotted onto the microarrayslide prior to the kinase reaction. Detection of phosphorylation wasperformed as described earlier in this Example except that instead of abiotin conjugated RC20 anti-phosphotyrosine antibody, a horse radishperoxidase (HRP) conjugated antibody was used (BD Biosciences, San Jose,Calif.) and thus secondary detection was not needed. In this case,fluorescence signal was generated using an Alexa Fluor® 488 Tyramide/TSAHRP substrate mediated fluorescence amplification kit for bettersensitivity (Invitrogen Corporation, Carlsbad, Calif.). After imagingthe fluorescence signals corresponding to detection of phosphotyrosine(see imaging details later in this example), the slides were furtherprobed with an anti-HSV antibody and fluorescent secondary antibody asdescribed in Example 5 to determine the amount of totalphoto-transferred protein based on their common C-terminal HSV epitopetags.

Detection of Fluorescence Signals:

All fluorescence signals were imaged on a FluorImager SI argonlaser-based scanner (Molecular Dynamics/Amersham Biosciences Corp.,Piscataway, N.J.).

Results:

Results are shown in FIG. 11. CK casein kinase II; MDM=ubiquitin-proteinligase E3 MDM2; p53=cellular tumor antigen p53; PKA=protein kinase Acatalytic subunit alpha; Tub=alpha-tubulin. FIG. 11A shows the minus ATPkinase reaction negative control, the phosphotyrosine positive controland the confirmation of successful contact photo-transfer. As expected,when only the needed ATP is omitted from the kinase reaction, nophosphorylation of the photo-transferred proteins is detected. However,the artificial pre-made and spotted phosphotyrosine-BSA conjugatepositive control clearly shows the antibody based phosphotyrosinedetection method works. Subsequent probing of the microarray slide withan anti-HSV antibody against the common HSV epitope tag present in allexpressed proteins confirms successful photo-transfer of all proteins,which are shown to be present in similar quantities on the arraysurface. FIG. 11B shows the results of the plus ATP kinase reaction for2 different human recombinant tyrosine kinases, ZAP-70 and Src pp⁶⁰. Theresults show differential phosphorylation of the variousphoto-transferred proteins (substrates) by the 2 kinases. Both kinasesheavily phosphorylate the MDM protein and to a lesser degreealpha-tubulin. However, Src pp⁶⁰ shows a broader substrate preference,with significant phosphorylation of CK and PKA. In contrast, ZAP-70 doesnot phosphorylate CK and phosphorylates PKA to a very slight, nearlyundetectable degree. For partial verification of the assay,alpha-tubulin was included as a substrate since it is known in theliterature to be phosphorylated by the ZAP-70 tyrosine kinase [Isakov etal. (1996) J Biol Chem 271, 15753-15761], and as expected, is indeedtargeted by the ZAP-70 kinase. Furthermore, as a negative control, p53is not phosphorylated by either kinase in correlation with the fact thatp53 is a major serine/threonine kinase substrate but not tyrosine kinasesubstrate.

Example 14 Contact Photo-Transfer from Individually Resolved Beads toMicroarray Surfaces Using Incorporated PC-Biotin: Detection of InternaltRNA Mediated Label and by Antibody

Cell-Free Expression and tRNA Mediated Labeling:

Human MDM2 and alpha-tubulin were expressed in a rabbit reticulocytecell-free reaction and co-translationally labeled with both BODIPY-FLand PC-biotin as in Example 1 with the following exceptions:BODIPY-FL-tRNA^(COMPLETE) was used for fluorescence labeling instead ofBODIPY-FL-tRNA^(Lys). As a negative control, an expression reaction wasperformed lacking only the added DNA for the gene of interest (Minus DNAblank). The Translation Dilution Buffer (TDB) used to stop the reactionand prepare the sample contained 0.2% (w/v) beta-casein (pure frombovine milk; Sigma-Aldrich, St. Louis, Mo.) instead of BSA as a carrier,10 mM DTT instead of 2 mM and additionally contained 20 mM EDTA addedfrom a 500 mM pH 8.0 stock and 4 mM cycloheximide (Sigma-Aldrich, St.Louis, Mo.) added from a 355 mM stock in DMSO.

Isolation of Labeled Nascent Proteins:

The isolation procedure only (see later for contact photo-transfer) wasperformed as in Example 1 with the following exceptions: Capture on theNeutrAvidin beads was for 30 min. After capture, beads were washed 2×briefly (briefly=5 sec vortex mix) with 45 bead volumes each of PBS pH7.5, 5 mM DTT and 0.1% (w/v) beta-casein and 2×5 min with 45 beadvolumes of 40% glycerol and 5 mM DTT in PBS (room temperature). Thewashed bead pellet was then prepared with 40% glycerol and 5 mM DTT inPBS to yield a 1% bead suspension (v/v).

Contact Photo-Transfer from Individually Resolved Beads:

For contact photo-transfer from individually resolved beads, the beadswere resuspended by mixing and 1 μL of the bead suspension was manuallypipetted onto the surface of a reactive epoxy activated glass microarraysubstrate (i.e. activated glass slide) (SuperEpoxy substrates, TeleChemInternational, Inc. ArrayIt™ Division, Sunnyvale, Calif.). Note that 1μL of the 1% bead suspension deposited on the substrate (so-called“parent spots”; ˜2 mm diameter) contained roughly 5 to 8 individualagarose beads (˜100 micron diameter) prior to removal/washing. Theindividual beads (prior to washing) within the parent spots were easilyvisible using a phase contrast light microscope and were typically notclustered/aggregated at this density (i.e. did not contact each other).Prior to photo-release, the beads were allowed to settle onto (contact)the microarray substrate surface by leaving the substrates for 5 minwithout disturbance/agitation. Note that in this buffer system, the moredense beads do visibly settle at unit gravity in this time frame. Thesubstrates were then illuminated, without agitation, for 5 min withnear-UV light (365 nm peak UV lamp, Blak-Ray Lamp, Model XX-15, UVP,Upland, Calif.) at a 5 cm distance to photo-release and transfer thetarget proteins. The power output of the lamp under these conditions was2.6 mW/cm² at 360 nm, 1.0 mW/cm² at 310 nm and 0.16 mW/cm² at 250 nm.After light treatment, the glass substrates were incubated withoutdisturbance for 30 min at 37° C. in a sealed and humidified chamber tofully ensure photo-released proteins react with the activated solidsurface. The beads and any unbound protein was then removed with 3×brief (5 sec) washes in TBS-T followed by 4× brief (5 sec) washes inpurified water. Phase contrast light microscopy reveals that the easilyvisible 100 micron NeutrAvidin agarose beads were completelywashed/removed from the glass substrates. The slides were dried prior tofluorescence imaging.

Detection of Photo-Transferred Protein:

Detection of the directly incorporated tRNA mediated BODIPY-FLfluorescence labeling was achieved by imaging the dry microarraysubstrates on an ArrayWoRx^(e) BioChip fluorescence reader (AppliedPrecision, LLC, Issaquah, Wash.) using the appropriate manufacturersupplied standard filter set and the resolution set to 9.7 microns.

Preparation of a Cy5 Conjugated Anti-HSV Antibody for FluorescenceDetection:

After imaging the signal from the directly incorporated tRNA mediatedBODIPY-FL fluorophores, the photo-transferred proteins on the microarraysubstrate were then probed with an antibody to the common C-terminal HSVepitope tag. For this, a fluorescently labeled Cy5 antibody conjugatewas prepared. 120 μg of mouse monoclonal anti-HSV tag antibody (EMDBiosciences, Inc., San Diego, Calif.) at 1 μg/μL was kept in it'smanufacturer supplied buffer (PBS/glycerol) and supplemented with1/9^(th) volume of 1M sodium bicarbonate stock for a final 100 mM. TheCy5—NHS monoreactive ester (Amersham Biosciences Corp., Piscataway,N.J.) labeling reagent was added to a 20-fold molar excess relative tothe antibody from a 27 mM stock prepared in DMSO. The reaction wasallowed to occur for 30 min with gentle mixing protected from light.Unreacted or hydrolyzed labeling reagent was removed using a NAP-10Sepharose G-25 desalting column (Amersham Biosciences Corp., Piscataway,N.J.) against a PBS buffer according to the manufacturer's instructionsexcept that only the visibly blue colored (Cy5) protein elution fractionwas collected. The antibody conjugate was analyzed in a standardspectrophotometer and found to be 0.07 mg/mL antibody concentration at 1mL total with an average of 3.5 Cy5 dyes per antibody molecule. Theantibody conjugate was then supplemented with a BSA carrier to a final0.1% (w/v) from a 10% stock and stored protected from light at +4° C.

Probing the Photo-Transferred Proteins with the Cy5 FluorescentlyLabeled Anti-HSV Antibody:

Microarray substrates were blocked for 15 min at 37° C. using 5% BSA(w/v) in TBS-T and then probed for 30 min at 37° C. with theanti-HSV-Cy5 probe at 0.7 ng/μL in the same buffer. Substrates were thenwashed 4×2 min with excess TBS-T, 4× briefly (5 sec) with purified waterand then dried. Detection of the anti-HSV-Cy5 signal was achieved byimaging the dry microarray substrates on an ArrayWoRx^(e) BioChipfluorescence reader using the appropriate manufacturer supplied standardfilter set (Applied Precision, LLC, Issaquah, Wash.) and the resolutionset to 9.7 microns. Results.

Results are shown in FIG. 12. −DNA=minus DNA blank derived fromexpression reaction lacking only the added DNA for gene of interest (allother processing steps otherwise performed same as with DNA containingexpressed protein samples); MDM=ubiquitin-protein ligase E3 MDM2;Tub=alpha-tubulin. The results show that the directly incorporatedBODIPY-FL fluorescence label is easily detectible following contactphoto-transfer of expressed proteins from individual 100 micron agarosebeads compared to the minus DNA blank. Individual spot diameters weremeasured using the manufacturer supplied software for the ArrayWoRx^(e)BioChip fluorescence reader (Applied Precision, LLC, Issaquah, Wash.)and were approximately 100 microns (note that agarose beads are suppliedmesh filtered and somewhat heterogeneous in size). Approximately 3-8individual spots originating from individual beads were observed withineach 1 μL (2 mm) parent spot in correlation with the number and patternof beads observed by phase contrast light microscopy prior to washingthe beads away. Prior to probing the microarrays with the anti-HSV Cy5antibody, the arrays were imaged using the Cy5 filter set (channel) inthe reader as a negative control. No cross-talk of the BODIPY-FLfluorescence from the tRNA mediated labels was observed in the Cy5filter set (channel) of the reader. Following probing the microarrayswith the anti-HSV Cy5 antibody, the arrays were again imaged with theCy5 filter set (channel) in the reader. Specific signal from thephoto-transferred HSV tagged proteins is clearly observed again withspotting patterns that precisely match that observed from the tRNAmediated fluorescence labeling. Clearly/sharply resolved and robust 100micron fluorescent spots suggests that the photo-released proteins arelargely captured/transferred directly onto the microarray surfacewithout significant diffusion into the fluid medium of the 1 μL parentspots.

Example 15 Contact Photo-Transfer from Individually Resolved Beads toMicroarray Surfaces Using Incorporated PC-Biotin: Advanced 2 Colorp53-MDM Protein-Protein Interaction Assays

Cell-Free Expression and tRNA Mediated Labeling:

The lower sample volume requirements per microarray-feature of themethod comprising contact photo-transfer from individually resolvedbeads facilitates significant scaling down of the expression reaction.Therefore the expression reaction was scaled down 10× compared toExample 1, from 200 μL to 20 μL. Note that with the isolation proceduresused (see later in this example) 20 μL of expression reaction yields 750agarose beads and therefore a theoretical maximum of 750 microarrayfeatures. Human MDM2 and GST were expressed in a rabbit reticulocytecell-free reaction and co-translationally labeled with both BODIPY-FLand PC-biotin as in Example 1 (scaled down proportionally to 20 μLreaction) with the following additional exceptions:

BODIPY-FL-tRNA^(COMPLETE) was used for fluorescence labeling instead ofBODIPY-FL-tRNA^(Lys). As a negative control, an expression reaction wasperformed lacking only the added DNA for the gene of interest (Minus DNAblank). The Translation Dilution Buffer (TDB) used to stop the reactionand prepare the sample contained 0.2% (w/v) beta-casein (pure frombovine milk; Sigma-Aldrich, St. Louis, Mo.) instead of BSA as a carrier,10 mM DTT instead of 2 mM and additionally contained 20 mM EDTA addedfrom a 500 mM pH 8.0 stock and 4 mM cycloheximide (Sigma-Aldrich, St.Louis, Mo.) added from a 355 mM stock in DMSO. Note that due to thescaled down reaction size, after TDB addition the total sample volumewas 40 μL prior subsequent steps.

Isolation of Labeled Nascent Proteins:

PC-biotin labeled nascent proteins were captured and isolated on 1 μLpacked bead volume of NeutrAvidin agarose beads having an approximatebiotin binding capacity of 80 pmoles (Pierce Biotechnology, Inc.,Rockford, Ill.). To facilitate addition of small bead volumes to thesamples, the beads were initially prepared to a 5% (v/v) bead suspensionin 0.1% (w/v) beta-casein, 1% (w/v) BSA and 5 mM DTT in PBS. Theprepared 40 μL of samples (see earlier in this Example) were then mixedwith 20 μL of the 5% (v/v) bead suspension corresponding to addition of1 μL packed bead volume. The isolation procedure was performed in batchmode using 0.45 micron pore size, PVDF membrane, micro-centrifugeFiltration Devices to facilitate manipulation of the small volumes ofaffinity matrix (˜100 micron beads) and exchange the buffers(Ultrafree-MC Durapore Micro-centrifuge Filtration Devices, 400 μLcapacity; Millipore, Billerica, Mass.). All steps were performed at +4°C. or on an ice water bath and all reagents and samples were also keptunder these conditions during the procedure. After capture on theNeutrAvidin beads for 30 min, beads were washed by mixing 2× briefly(briefly=5 sec vortex mix) and 2× for 5 min in 400 bead volumes perwash. The buffer used for washing the beads was PBS pH 7.5 and 5 mM DTT.The beads were then additionally washed 1× briefly (briefly=5 sec vortexmix) in 400 bead volumes of 40% glycerol and 5 mM DTT in PBS. Prior tocontact photo-transfer of the captured and isolated proteins, the washedpellet of 1 μL of NeutrAvidin agarose beads was suspended in a finalvolume of 100 μL of 40% glycerol and 5 mM DTT in PBS thereby resultingin a 1% (v/v) bead suspension that can be stored long-term at −20° C.without freezing of the sample and thus without damage to theNeutrAvidin agarose beads.

Contact Photo-Transfer from Individually Resolved Beads:

Performed as in Example 14 with the following exceptions: 0.5 μL insteadof 1 μL of the 1% (v/v) bead suspension was applied to the microarraysubstrates to create the parent spots and application was to aldehydeactivated glass microarray substrates instead of epoxy (i.e. activatedglass slide) (SuperAldehyde substrates, TeleChem International, Inc.ArrayIt™ Division, Sunnyvale, Calif.). Note that 0.5 μL of the 1% beadsuspension deposited on the substrate (so-called “parent spots”; ˜1-2 mmdiameter) contained roughly 3 to 5 individual agarose beads (˜100 microndiameter) prior to removal/washing. After contact photo-transfer fromindividually resolved beads, the beads and any unbound protein were thenremoved and the substrates simultaneously blocked with a 15 min wash,with mixing, using 5 mM DTT, 100 mM glycine and 6% (w/v) BSA in PBS.Phase contrast light microscopy reveals that the easily visible 100micron NeutrAvidin agarose beads were completely washed/removed from theglass substrates.

Preparation of a Cy5 Conjugated p53 Probe for Fluorescence Detection ofProtein-Protein Interaction:

In order to measure the known biological binding interaction between themicroarray deposited MDM “bait” and p53, a fluorescently labeled p53-Cy5conjugate/probe was prepared as follows: 100 μL of a commerciallyavailable recombinant human p53-GST fusion protein (Santa CruzBiotechnology, Inc., Santa Cruz, Calif.) at 1 μg/μL (100 μg) was usedfor fluorescence labeling. The manufacturer supplied p53 solution wasclarified in a micro-centrifuge at 13,000 rpm for 5 min. The p53 wasthen dialyzed against 200 mM sodium bicarbonate, 200 mM NaCl, 5 mM DTTand 10 mM EDTA (added from a 500 mM pH 8.0 stock). Dialysis wasperformed at +4° C. in 400 μL capacity 10 kDa molecular weight cut-off(MWCO) Slide-A-Lyzer MINI Dialysis Units (Pierce Biotechnology, Inc.,Rockford, Ill.). Reservoir buffer was 250-500 mL for each round ofdialysis and dialysis was for 1× overnight and 1×1 hr with mixing of thereservoir buffer using a standard magnetic stir bar device. Theresultant dialyzed p53 sample is collected and mixed with 0.5 μL of a 27mM stock of Cy5—NHS monoreactive ester (Amersham Biosciences Corp.,Piscataway, N.J.) which was prepared in DMSO. With an estimated 50%protein recovery after dialysis, this constitutes an approximate 20-foldmolar excess of labeling reagent. The labeling reaction was allowed toproceed by gentle mixing for 30 min protected from light with aluminumfoil. 1/9^(th) volume freshly prepared 100 mM L-lysine monohydrochloridein PBS was added to quench the reaction and mixed for 1 hr protectedfrom light with aluminum foil. After quenching, the sample is mixed withequal volume of 10 mM DTT and 0.2% (w/v) beta-casein in 2×PBS prior toprocessing on a desalting column. Unreacted or hydrolyzed labelingreagent was removed using a NAP-10 Sepharose G-25 desalting column(Amersham Biosciences Corp., Piscataway, N.J.) against 5 mM DTT and 0.1%(w/v) beta-casein in PBS according to the manufacturer's instructionsexcept that only the visibly blue colored (Cy5) protein elution fractionwas collected. Estimated p53-Cy5 conjugate concentration is 25-50 ng/μL(protein concentration). Note that initially, a similar p53 labelingprocedure was done except using a BODIPY-FL-SSE labeling reagent(Invitrogen Corporation, Carlsbad, Calif.) instead of the Cy5—NHSmonoreactive ester. This allowed analysis of the conjugate via SDS-PAGEand fluorescence imaging of the gel using a FluorImager SI argonlaser-based scanner (Molecular Dynamics/Amersharn Biosciences Corp.,Piscataway, N.J.) in order to verify successful conjugation and estimateprotein concentration using known standards. This p53-Cy5 probe stockwas stored in single to double use aliquots at −70° C.

Probing the Photo-Transferred Proteins with the Cy5 FluorescentlyLabeled p53 Probe:

The MDM and GST proteins were immobilized/transferred onto themicroarray substrates which were then washed and blocked as describedearlier in this Example. The microarray substrates were subsequentlyprobed with the p53-Cy5 conjugate to test for its expected biologicalinteraction with MDM. To do so, microarray substrates were first furtherwashed 3× briefly (5 sec) with excess TBS. The p53-Cy5 probe stockprepared as described earlier in this Example was thawed and diluted 1:1with 10% BSA (w/v) and 5 mM DTT and further supplemented with 1/49^(th)volume of a 5M NaCl stock. The final buffer composition of the dilutedp53-Cy5 probe was 5% BSA (w/v), 150 mM NaCl, 0.05% beta-casein (w/v) and5 mM DTT in 25 mM sodium phosphate pH 7.5. Insoluble, aggregated orparticulate material/contamination was removed from the probe solutionby running it through a 0.45 micron pore size, PVDF membrane,micro-centrifuge Filtration Device (Ultrafree-MC DuraporeMicro-centrifuge Filtration Devices, 400 μL capacity; Millipore,Billerica, Mass.) in addition to further spinning the filtrate in amicro-centrifuge at 13,000 rpm and collecting the fluid supernatant.Each microarray substrate was probed with ˜75 μL of the diluted p53-Cy5probe solution by overlaying with a 22×60 mm glass coverslip andincubating for 30 min in a humidified chamber. Unbound probe was thenremoved by washing 3×1 min each with excess PBS and then 1× briefly (5sec) purified water. The microarray substrates were then dried.

Detection of Photo-Transferred Protein:

Detection of the directly incorporated tRNA mediated BODIPY-FLfluorescence labeling as well as binding of the fluorescent p53-Cy5probe was achieved by imaging the dry microarray substrates on anArrayWoRx^(e) BioChip fluorescence reader (Applied Precision, LLC,Issaquah, Wash.) using the appropriate standard built-in filter sets todiscriminate between the 2 color fluorophores and the resolution set to9.7 microns.

Results:

Results are shown in FIG. 13. −DNA=minus DNA blank derived fromexpression reaction lacking only the added DNA for gene of interest (allother processing steps otherwise performed same as with DNA containingexpressed protein samples); MDM=ubiquitin-protein ligase E3 MDM2;GST=glutathione-s-transferase. The results show that the directlyincorporated BODIPY-FL fluorescence label is easily detectible followingcontact photo-transfer of expressed proteins from individual ˜100 micronagarose beads compared to the minus DNA blank. Individual spot diameterswere measured using the manufacturer supplied software for theArrayWoRx^(e) BioChip fluorescence reader (Applied Precision, LLC,Issaquah, Wash.) and were approximately 100 microns. The arrow in FIG.13 denotes a single spot derived from the contact photo-transfer fromindividually resolved beads that was specifically measured at 100microns in diameter. Four individual spots originating from individualbeads were observed within each 0.5 μL (1-2 mm) parent spot incorrelation with the number and pattern of beads observed by phasecontrast light microscopy prior to washing the beads away. Query of themicroarray substrate with the p53-Cy5 probe clearly shows the probe onlyinteracts with MDM as expected, in correlation with the literature[Bottger et al. (1997) J Mol Biol 269, 744-756], and not the GSTnegative control protein present in equal amounts based on the BODIPY-FLsignals. Clearly/sharply resolved and robust 100 micron fluorescentspots suggests that the photo-released proteins are largelycaptured/transferred directly onto the microarray surface withoutsignificant diffusion into the fluid medium of the 0.5 μL parent spots.

Example 16 Contact Photo-Transfer from Individually Resolved Beads ofPre-Formed Protein-Protein Complexes to Microarray Surfaces UsingIncorporated PC-Biotin: Protein-Protein Interaction Assays Using OnlyCell-Free Expressed and tRNA Labeled Proteins Throughout

Cell-Free Expression and tRNA Mediated Labeling:

The lower sample volume requirements per microarray-feature of themethod comprising contact photo-transfer from individually resolvedbeads facilitates significant scaling down of the expression reaction.Therefore the expression reaction was scaled down 10× compared toExample 1, from 200 μL to 20 μL. Note that with the isolation proceduresused (see later in this example) 20 μL of expression reaction yields˜750 agarose beads and therefore a theoretical maximum of 750 microarrayfeatures. Human MDM2, GST and p53 were expressed in a rabbitreticulocyte cell-free reaction and co-translationally labeled withBODIPY-FL or PC-biotin as in Example 1 (scaled down proportionally to 20μL reaction) with the following additional exceptions:

BODIPY-FL-tRNA^(COMPLETE) was used for fluorescence labeling (at 2 μM)instead of BODIPY-FL-tRNA^(Lys). The cell-free expressed p53 “probe” waslabeled only with BODIPY-FL using the BODIPY-FL-tRNA^(COMPLETE) and wasnot labeled with PC-biotin in any way. Note that 40 μL of p53 wasexpressed and processed such that 20 μL could be used to probe each ofthe 2 “bait” proteins (MDM2 and GST). The “bait” proteins were labeledonly with PC-Biotin-tRNA^(COMPLETE) (at 1 μM) and not with BODIPY-FL inany way. The Translation Dilution Buffer (TDB) used to stop the reactionand prepare the sample contained no BSA or other protein carrier, 10 mMDTT instead of 2 mM and additionally contained 20 mM EDTA added from a500 mM pH 8.0 stock and 4 mM cycloheximide (Sigma-Aldrich, St. Louis,Mo.) added from a 355 mM stock in DMSO. Note that due to the scaled downreaction size, after TDB addition the total sample volume was 40 μL (for“bait” proteins) prior subsequent steps (80 μL for p53 “probe”).

Protein-Protein Interaction Assay:

All steps were performed at +4° C. or on an ice water bath and allreagents and samples were also kept under these conditions during theprocedure. 40 μL of the processed/diluted p53 “probe” solution was mixedwith each of the 40 μL of the processed/diluted “bait” proteins (MDM2and GST) and incubated for 15 min at +4° C. with gentle mixing to allowany binding to occur. PC-biotin labeled “bait” proteins (MDM2 and GST),along with any bound BODIPY-FL labeled p53 “probe”, were then capturedand isolated on 1 μL packed bead volume of NeutrAvidin agarose beadshaving an approximate biotin binding capacity of 80 pmoles (PierceBiotechnology, Inc., Rockford, Ill.). To facilitate addition of smallbead volumes to the samples, the beads were initially prepared to a 10%(v/v) bead suspension with 5 mM DTT in PBS. The samples, now 80 μL each,were then mixed with 10 μL of the 10% (v/v) bead suspensioncorresponding to addition of 1 μL packed bead volume. After capture onthe NeutrAvidin beads for 1 hr, beads were washed by mixing 3× briefly(briefly=5 sec vortex mix) in 400 bead volumes per wash of PBS pH 7.5and 5 mM DTT. The washing procedure was performed in batch mode using0.45 micron pore size, PVDF membrane, micro-centrifuge FiltrationDevices to facilitate manipulation of the small volumes of affinitymatrix (˜100 micron beads) and exchange the buffers (Ultrafree-MCDurapore Micro-centrifuge Filtration Devices, 400 μL capacity;Millipore, Billerica, Mass.). Prior to contact photo-transfer of thecaptured and isolated proteins, the washed pellet of 1 μL of NeutrAvidinagarose beads was suspended in a final volume of 50 μL of 50% glyceroland 5 mM DTT in PBS thereby resulting in a 2% (v/v) bead suspension.

Contact Photo-Transfer from Individually Resolved Beads:

Performed as in Example 14 except that after contact photo-transfer fromindividually resolved beads to epoxy activated microarray substrates,the beads and any unbound protein were then removed by washing only 1×briefly (5 sec) in purified water. Phase contrast light microscopyreveals that the easily visible 100 micron NeutrAvidin agarose beadswere completely washed/removed from the glass substrates. Substrateswere dried prior to fluorescence imaging.

Detection of Protein-Protein Interaction After Contact Photo-Transfer:

Detection of the directly incorporated tRNA mediated BODEPY-FLfluorescence signal arising from selective binding of the cell-freeexpressed p53 “probe” to the MDM “bait” was achieved by imaging the drymicroarray substrates on an ArrayWoRx^(e) BioChip fluorescence reader(Applied Precision, LLC, Issaquah, Wash.) using the appropriate standardbuilt-in filter set.

Detection of Total Photo-Transferred Protein Using an Anti-HSV-Cy5Antibody:

After determining binding of the p53 “probe” by fluorescence imaging,the successful photo-transfer of all proteins was verified using ananti-HSV-Cy5 fluorescently labeled antibody against the commonC-terminal HSV epitope tag present in all expressed proteins asdescribed in Example 14.

Results:

Results are shown in FIG. 14. MDM=ubiquitin-protein ligase E3 MDM2 (“×4”refers to application of 4 parent spots at 1 μL each);GST=glutathione-s-transferase (“×3” refers to application of 3 parentspots at 1 μL each). The results show that the directly incorporatedBODIPY-FL fluorescence label corresponding to selective binding of thep53 “probe” to the MDM “bait”, in correlation with the literature[Bottger et al. (1997) J Mol Biol 269, 744-756], is easily detectiblefollowing contact photo-transfer of the expressed/isolated proteins andcomplexes from individual ˜100 micron agarose beads. In contrast, theGST negative control “bait” shows no signal indicating no binding of theadded p53 “probe” as expected. Note that in comparison to Examples 14and 15, the beads were 2× more concentrated and thus somewhat clusteredwithin the parent spots and therefore not all beads are fully resolvedwith this lower magnification image (although many are). Further queryof the microarray substrate with the anti-HSV-Cy5 antibody against thecommon C-terminal epitope tag present in all expressed proteins clearlyshows that both the MDM and GST “baits” were successfullyphoto-transferred although only the MDM binds the p53 “probe”. Note thatGST expresses more efficiently than p53 or MDM and therefore provides astronger anti-HSV-Cy5 signal than even the p53-MDM complexes.

Example 17 Contact Photo-Transfer to Activated Microarray Surfaces UsingPhotocleavable Antibodies: Detection of a tRNA Mediated DirectFluorescence Label Preparation of a Photocleavable Antibody AffinityMatrix:

For photo-isolation of expressed proteins, an antibody against thecommon C-terminal HSV epitope tag was conjugated to PC-biotin and loadedto a NeutrAvidin agarose bead affinity matrix as done in Example 2.

Cell-Free Expression and tRNA Mediated Labeling:

Human p53 oncoprotein (tumor antigen) and glutathione-s-transferase(GST) proteins containing a common HSV epitope tag at the C-terminuswere expressed and labeled in a rabbit reticulocyte cell-free reactionsystem as described in Example 1 except that onlyBODIPY-FL-tRNA^(COMPLETE) was used for labeling at 1 μM and the BSAprotein carrier was omitted from the TDB buffer used to stop thereaction and prepare the sample.

Isolation of Labeled Nascent Proteins:

The isolation procedure only (see later for contact photo-transfer andsolution photo-release) was performed as in Example 1 with the followingexceptions: 20 μL of the anti-HSV photocleavable antibody affinitymatrix was substituted for the NeutrAvidin beads in Example 1. Thebuffers used in the procedure contained no BSA or other protein carriersat any step. 50 bead volumes per wash was used to remove the unboundmaterial. The washed bead pellet was then suspended to a 50% bead slurry(v/v) in 40% glycerol and 1 mM DTT in PBS.

Contact Photo-Transfer:

Performed as in Example 7 except that amine-reactive aldehyde activatedglass microarray substrates (i.e. activated glass slide) (SuperAldehydesubstrates, TeleChem International, Inc. ArrayIt™ Division, Sunnyvale,Calif.) were used instead of the epoxy activated substrates.

Solution Photo-Release for SDS-PAGE Analysis:

For quality control confirmation, the remaining sample/beads (50%suspension) not used for contact photo-transfer were diluted to anapproximate 10% bead suspension (v/v) in 0.1% BSA (w/v) and 1 mM DTT inPBS. Photo-release of this bead suspension and SDS-PAGE analysis wasperformed as described in Example 1. An aliquot of the crudenon-isolated cell-free expression reaction was also analyzed in parallelvia standard SDS-PAGE.

Detection of Proteins:

Detection of the directly incorporated tRNA mediated BODIPY-FLfluorescence labeling was achieved by imaging either the dry microarraysubstrates or the electrophoretic gel on a FluorImager SI laser-basedscanner (Molecular Dynamics/Amersham Biosciences Corp., Piscataway,N.J.).

Results:

Results are shown in FIG. 15. PC-Antibody=photocleavable HSV antibodyisolated fractions; Crude=crude non-isolated cell-free expressionreaction (equivalent loading to PC-Antibody fractions); +hν=elution(photo-release) with proper light illumination; −hν=elution procedurewithout light illumination; −DNA=minus DNA blank derived from expressionreaction lacking only the added DNA for gene of interest (all otherprocessing steps otherwise performed same as with DNA containingexpressed protein samples); GST=glutathione-s-transferase; p53=cellulartumor antigen p53. FIG. 15A shows the fluorescence SDS-PAGE bandscorresponding to the GST and p53 proteins at the correct approximatemolecular weight positions. The highly fluorescent unresolved zone atthe bottom of the gel in the crude samples corresponds to unusedfluorescence tRNA and byproducts as well as auto-fluorescence from largequantities of hemoglobin in the rabbit reticulocyte cell-free expressionlysate. The photo-release lanes show recovery of the purified proteinsonly when the appropriate light illumination is used, with onlynegligible trace quantities “leached” from the affinity matrix in theabsence of light illumination. FIG. 15B shows the contact photo-transferto an aldehyde activated microarray substrate. The internal tRNAmediated fluorescence label is clearly detectible in the transferredproteins with signal to noise ratios of 10:1 and 8:1 for GST and p53respectively.

Example 18 Photo-Transfer to Nickel Metal Chelate Coated MicrotiterPlates Using Photocleavable Antibodies: Detection of the Already-BoundPhotocleaved Antibody Preparation of a Photocleavable Antibody AffinityMatrix:

For photo-isolation of expressed proteins, an antibody against thecommon C-terminal HSV epitope tag was conjugated to PC-biotin and loadedto a NeutrAvidin agarose bead affinity matrix as done in Example 2.

Cell-Free Expression and tRNA Mediated Labeling:

Human casein kinase II (CK) and human dihydrofolate reductase (DHFR)proteins containing a common HSV and polyhistidine epitope tag at theC-terminus were expressed in a rabbit reticulocyte cell-free reactionsystem as described in Example 1 except that no misaminoacylated tRNAswere used (no labeling), the expression was carried out for 1 hr and theBSA protein carrier was omitted from the TDB buffer used to stop thereaction and prepare the sample.

Isolation of Labeled Nascent Proteins:

The isolation procedure only (see later for photo-transfer) wasperformed as in Example 1 with the following exceptions: 20 μL of theanti-HSV photocleavable antibody affinity matrix was substituted for theNeutrAvidin beads in Example 1. The buffers used in the procedurecontained no BSA or other protein carriers at any step. 50 bead volumesper wash was used to remove the unbound material. The washed bead pelletwas then suspended to a 50% bead slurry (v/v) in 40% glycerol and 1 mMDTT in PBS.

Photo-Transfer to Wells of a Nickel Metal Chelate Coated MicrotiterPlate:

The 50% bead slurry corresponding to the captured protein samples wasfurther diluted to a 2.5% bead suspension in TBS-T and loaded at 100μL/well to commercially available nickel metal chelatecoated/derivatized opaque white 96-well microtiter plates (PierceBiotechnology, Inc., Rockford, Ill.). Photo-transfer from the affinitybeads to the wells of the microtiter plate was achieved as in Example 10except that the capture mechanism onto the plate was via the C-terminalpolyhistidine tag present in the photo-released protein and the capturestep was allowed to occur for 30 min.

Detection of Photo-Transferred Protein:

Following photo-transfer of the target proteins to the metal chelatecoated microtiter plate wells, the bead suspension was removed and thewells washed 4× briefly (5 sec) in 300 μL/well of TBS-T. Detection ofthe already-bound HSV mouse monoclonal antibody (from the photocleavableantibody isolation step) was achieved using a secondary rabbitanti-[mouse IgG] horseradish peroxidase (HRP) conjugate (PierceBiotechnology, Inc., Rockford, Ill.). The detection antibody was addedat a 1/50,000 dilution of the manufacturer's stock in 1% BSA (w/v) inTBS-T for 30 min. Plates were washed again and signal was generatedusing a commercially available chemiluminescence HRP substrate(SuperSignal Femto ELISA Substrate; Pierce Biotechnology, Inc.,Rockford, Ill.) according to the manufacturer's instructions. Signal wasread in a LumiCount luminescence plate reader (Packard/PerkinElmer Lifeand Analytical Sciences, Inc., Boston, Mass.).

Results:

Results are shown in FIG. 16. −DNA=minus DNA blank derived fromexpression reaction lacking only the added DNA for gene of interest (allother processing steps otherwise performed same as with DNA containingexpressed protein samples); GST=glutathione-s-transferase;DHFR=dihydrofolate reductase; RLU=raw relative luminescence units. Theresults show clear detection of the photo-transferred GST and DHFR withsignal to noise ratios of 108:1 and 19:1 respectively compared to theminus DNA negative control sample. It is important to note that like theexpressed protein samples, the minus DNA control would also containphoto-released anti-HSV antibody that is not bound to any expressedprotein. Therefore, the results demonstrate that the specific signalachieved for the GST and DHFR proteins is indeed a result of detectionof only photo-released anti-HSV antibody that is bound to the expressedproteins which are in turn themselves bound to the nickel metal chelatecoated plate via their polyhistidine tag proteins have HSV andpolyhistidine tags); and any photo-released anti-HSV antibody not boundto it's target protein is effectively washed out of the wells of theplate since it lacks any metal chelate binding tag.

Example 19 Contact Photo-Transfer to Activated Microarray Surfaces UsingPhotocleavable Antibodies: Application to Advanced 2 ColorProtein-Protein Interaction Assays

Cell-Free Expression and tRNA Mediated Labeling

Various human proteins containing a common HSV epitope tag at theC-terminus were expressed in a rabbit reticulocyte cell-free reactionand co-translationally labeled with BODIPY-FL as in Example 1 with thefollowing exceptions: BODIPY-FL-tRNA^(COMPLETE) was used forfluorescence labeling instead of BODIPY-FL-tRNA^(Lys).PC-Biotin-tRNA^(COMPLETE) was not used for direct labeling sinceisolation was via a photocleavable antibody instead (see later in thisExample). As a negative control, an expression reaction was performedlacking only the added DNA for the gene of interest (Minus DNA blank).The Translation Dilution Buffer (TDB) used to stop the reaction andprepare the sample contained no BSA or any other protein carriers.

Preparation of a Photocleavable Antibody Affinity Matrix:

An anti-HSV tag photocleavable antibody conjugated agarose bead affinitymatrix was prepared as in Example 2.

Isolation of Labeled Nascent Proteins:

The isolation procedure only (see later for contact photo-transfer) wasperformed as in Example 1 with the following exceptions: The buffersused in the procedure contained no BSA or other protein carriers at anystep. Capture was on 10 μL of the anti-HSV photocleavable antibodybeads. After washing the unbound material from the NeutrAvidin beads asdescribed in Example 1 the beads were further washed 3× briefly(briefly=5 sec vortex mix) with 45 bead volumes each of plain PBS and1×5 min with 45 bead volumes of 40% glycerol in PBS. The washed beadpellet was then suspended with equal volume of 40% glycerol in PBS toyield a 50% bead slurry (v/v).

Contact Photo-Transfer:

Performed as in Example 7 except that some protein samples (GST, p53 andTub) were further diluted with unused/plain anti-HSV photocleavableantibody beads (beads still in 40% glycerol and PBS) to decrease thetotal integrated amount of transferred protein in the applied 1 μL (˜2mm) parent spot to a level roughly similar (although not exact) to thelower expressing and poorer substrate binding Calm and TNF proteins.

Preparation of a Calcineurin-Cy5 Directly Labeled Fluorescence Probe:

In order to measure the known biological binding interaction between themicroarray deposited calmodulin “bait” and calcineurin, a fluorescentlylabeled calcineurin-Cy5 conjugate/probe was prepared as described inExample 12.

Probing the Proteins on the Microarray with the Calcineurin-Cy5Conjugate:

The cell-free expressed proteins subsequently photo-transferred onto themicroarray substrates as described earlier in this Example were thenprobed with the calcineurin-Cy5 conjugate to test for its expectedbiological interaction with calmodulin. This was done as described inExample 12 except that no minus calcium permutation was performed.

Detection of Photo-Transferred Protein:

Detection of the directly incorporated tRNA mediated BODIPY-FLfluorescence labeling as well as binding of the fluorescentcalcineurin-Cy5 probe was achieved by imaging the dry microarraysubstrates on an ArrayWoRx^(e) BioChip fluorescence reader (AppliedPrecision, LLC, Issaquah, Wash.) using the appropriate standard built-infilter sets to discriminate between the 2 color fluorophores.

Results:

Results are shown in FIG. 17. −DNA=minus DNA blank derived fromexpression reaction lacking only the added DNA for gene of interest (allother processing steps otherwise performed same as with DNA containingexpressed protein samples); Calm=calmodulin;GST=glutathione-s-transferase; p53=cellular tumor antigen p53;Tub=alpha-tubulin; TNF=tumor necrosis factor alpha. The direct tRNAmediated BODIPY-FL labeling confirms that all proteins are present onthe array surface in amounts equal to or greater than the amount of thecalmodulin “bait”. It is important to note that the GST, p53 and Tubprotein samples were further diluted with unused agarose beads prior tophoto-transfer to roughly normalize the total integrated amount oftransferred proteins in the 1 μL (˜2 mm) parent spot. Importantly, inthose pre-diluted samples, the beads are dispersed sufficiently thattransfer from individual ˜100 micron agarose beads can be resolved(speckled appearance of parent spot), again supporting that afterphoto-release, the proteins are directly transferred due to the closebead-surface contact and that diffusion of the proteins away from thebead area is minimal. Furthermore, as expected, the calcineurin-Cy5probe interacts only with the calmodulin spots on the microarraysurface, in correlation with the known biological interaction asreported in the literature [Nakamura et al. (1992) FEBS Lett 309,103-106], and not with the other non-calmodulin binding proteins.

Example 20 Preparation of PC-Biotin Conjugated Quantum Dot Nanocrystalsand Binding to NeutrAvidin Agarose Beads Preparation of PC-BiotinConjugated Quantum Dot 605 Nanocrystals:

Antibody (IgG) conjugated Quantum Dot nanocrystals were obtainedcommercially (QDot® 605 Sheep anti-Digoxigenin Conjugate [Fab Fragment]catalog number 1600-1; Quantum Dot Corp., Hayward, Calif.) and wereprovided from the manufacturer at 1 μM concentration in borate buffer pH8.3. The binding specificity of the Quantum Dot conjugated antibody(IgG), against digoxigenin, was irrelevant in this case since the smallmolecule antigen digoxigenin occurs naturally only in plants. Theantibody (IgG) coating in this case served only as an irrelevant proteinmedium in order to mediate conjugation of PC-biotin using AmberGen'sproprietary protein/amine reactive PC-biotin NHS ester labeling reagent.Quantum Dots conjugated to other irrelevant proteins such as BSA orsimply amine derivatized (also available from Quantum Dot Corp.,Hayward, Calif.) would also be suitable. A 2 mM stock of the PC-biotinNHS ester labeling reagent was prepared in anhydrous dimethyl formamide(DMF) and 1 μL added to 100 μL of the manufacturer supplied Quantum Dotsfor an approximate 20-fold molar excess labeling reagent relative to theQuantum Dots. The reaction was allowed to proceed for 30 min with gentlemixing. Unreacted or hydrolyzed labeling reagent was removed using aNAP-10 Sepharose G-25 desalting column (Amersham Biosciences Corp.,Piscataway, N.J.) against a TBS buffer according to the manufacturer'sinstructions except that only the visibly orange colored (Quantum Dot)size-excluded elution fraction was collected. The resultant PC-biotinQuantum Dot conjugate was analyzed on a standard spectrophotometer andthe yielded Quantum Dot concentration calculated to be 0.17 μM at ˜500μL total (85% recovery) using the appropriate extinction coefficient.

Selective Binding of PC-Biotin Conjugated Quantum Dot 605 Nanocrystalsto NeutrAvidin Beads:

PC-biotin conjugated Quantum Dots were selectively captured and isolatedon 10 μL packed bead volume of NeutrAvidin agarose beads (˜100 microndiameter) having an approximate total biotin binding capacity of 800pmoles (Pierce Biotechnology, Inc., Rockford, Ill.). The isolationprocedure was performed in batch mode using a micro-centrifuge andpolypropylene tubes to manipulate the affinity matrix (beads) andexchange the buffers (note that under the micro-centrifuge conditionsused, ˜10 sec at 13,000 rpm, the Quantum Dots do not precipitate butremain in solution). Beads were first pre-washed 2× briefly (briefly=5sec vortex mix) with 45 bead volumes per wash using 5 mM DTT and 0.01%(w/v) Triton X-100 detergent in PBS. The beads were suspended with 100μL of the same buffer and 24 μL of the prepared 0.17 μM PC-biotinconjugated Quantum Dots was added, therefore constituting an approximatetheoretical maximum 1 to 2% level of the total available bindingcapacity of the 10 μL of NeutrAvidin beads (assuming that for stericreasons, a roughly 1:1 binding ratio of NeutrAvidin tetramer to QuantumDots, which are the size of large proteins, will occur). 1-2% ofsaturation was used since the Quantum Dots are anticipated to beultimately employed for photo-transferable spectral bar-coding of theNeutrAvidin beads, and the remaining binding capacity of the NeutrAvidinbeads will be needed for capture of other PC-biotin conjugatedbiomolecules (e.g. cell-free expressed proteins described in previousExamples). Additionally, as a negative control (blank), a parallelsample was performed but by adding the same amount of plain QuantumDots, i.e. not conjugated to PC-biotin but otherwise identical. Bindingwas allowed to occur for 30 min at +4° C. with gentle shaking.

Prior to washing away the unbound Quantum Dots from the NeutrAvidinagarose beads, the bead suspension was imaged directly in the clearpolypropylene micro-centrifuge tubes using a FluorImager SI argonlaser-based fluorescence scanner (Molecular Dynamics/AmershamBiosciences Corp., Piscataway, N.J.) and the standard manufacturersupplied 610 nm emissions filter. Unbound Quantum Dots where thenremoved from the NeutrAvidin agarose beads by washing the beads 3×briefly (briefly=5 sec vortex mix) and 1×1 hr (+4° C.) at 45 beadvolumes per wash using 5 mM DTT and 0.01% (w/v) Triton X-100 detergentin PBS. The washed bead pellet was imaged using a FluorImager SI argonlaser-based fluorescence scanner (Molecular Dynamics/AmershamBiosciences Corp., Piscataway, N.J.) and the standard manufacturersupplied 610 nm emissions filter.

Results:

Results are shown in FIG. 18. FIG. 18A shows the NeutrAvidin beadsuspension prior to washing away the unbound Quantum Dots. Thefluorescence signal arising from the total amount of added Quantum Dotsis the same for both the plain non-PC-biotin and the PC-biotinconjugated Quantum Dots as expected. FIG. 18B shows the NeutrAvidin beadpellets only, after washing away the unbound Quantum Dots. Significantbinding of the Quantum Dots only occurs in the case where they areconjugated to PC-biotin, with a 6-fold greater signal intensity than forthe plain non-PC-biotin Quantum Dots. The background signal in the plainnon-PC-biotin scenario does not arise from non-specifically boundQuantum Dots (as confirmed later in Example 21), but is the typicalbackground fluorescence from plain untreated NeutrAvidin agarose beads(not shown in FIG. 18) likely due to auto-fluorescence and lightscattering effects.

Example 21 Contact Photo-Transfer of Photocleavable Quantum DotNanocrystals to Activated Microarray Substrates: UV Dependence ofTransfer and Fluorescence Specificity Contact Photo-Transfer ofPC-Biotin Conjugated Quantum Dot Nanocrystals:

The same 10 μL NeutrAvidin agarose beads loaded with PC-biotinconjugated Quantum Dots prepared as described in Example 20 were furtherwashed 1× briefly (briefly=5 sec vortex mix) at 45 bead volumes with 5mM DTT and 40% glycerol in PBS and resuspended to a 50% (v/v) slurrywith the same buffer. Negative control beads treated with the sameamount of plain Quantum Dots, i.e. not conjugated to PC-biotin asdescribed in Example 20, were also processed in this way. Using thesebead slurrys, contact photo-transfer was performed as described inExample 7 with the following exceptions: As a negative control,additional spots of bead slurry that were applied to the same microarraysubstrate were not illuminated with the appropriate near-UV light byemploying shielding with an opaque aluminum foil covered barrier. Afterbinding of the transferred material to the microarray substrate, thesubstrates were washed 4×1 min with excess TBS-T and 4× briefly (5 sec)with purified water prior to drying and imaging.

Detection of Photo-Transferred Quantum Dots:

Detection of the Quantum Dot 605 nm fluorescence emissions was achievedby imaging the dry microarray substrates on an ArrayWoRx^(e) BioChipfluorescence reader (Applied Precision, LLC, Issaquah, Wash.) using thestandard built-in (manufacturer supplied) filter sets. The Cy3 filterset was used here to selectively image the Quantum Dot fluorescence.Although not optimal, these particular Quantum Dots (˜400 nm optimal butbroad excitation and 605 nm emissions peak) can be imaged using astandard Cy3 excitation-emissions filter set, albeit with 5× less signalintensity than Quantum Dot optimized fluorescence filters.

Results:

Results are shown in FIG. 19. “QDot”=refers to the procedure performedon plain Quantum Dots, i.e. not conjugated to PC-biotin; “PCB-QDot”refers to the procedure performed on PC-biotin (PCB) conjugated QuantumDots; “Channel”=refers to the various fluorescence filter sets used toobtain images; “UV”=the near-UV light illumination required forphotocleavage of the PC-biotin. FIG. 19 shows fluorescence imagesobtained by scanning the microarray substrates in the ArrayWoRx^(e)BioChip fluorescence reader (Applied Precision, LLC, Issaquah, Wash.),whereby signal from the photo-transferred Quantum Dot 605 nm emissionsis only expected with the Cy3 filter set (channel). As anticipated, whenattempts are made to load plain non-PC-biotin Quantum Dots toNeutrAvidin agarose beads, no binding occurs (Quantum Dots washed awayfrom beads in isolation step) and thus no measurable photo-transferoccurs from the beads to the microarray substrate. However, whenPC-biotin conjugated Quantum Dots are loaded to the NeutrAvidin agarosebeads, binding does occur as demonstrated previously in Example 20, andthus photo-transfer does occur as shown in FIG. 19. Fluorescence signalis only observed in the Cy3 channel as expected and no fluorescencecross-talk occurs in the “BODIPY-FL & Fluorescein” channel or the “Cy5”channel. As an additional negative control, when illumination with theproper near-UV light is not done, transfer of the PC-biotin conjugatedQuantum Dots from the NeutrAvidin agarose beads to the microarraysubstrate does not occur and signal is not observed in any fluorescencechannels (Cy3 channel is shown in FIG. 19). Note that the high densityof beads per 1 μL parent spot on the substrates does not afford goodresolution of photo-transfer from individual beads at this magnification(although speckled appearance indicates individual beads). Nonetheless,an example involving contact photo-transfer of Quantum Dots fromindividual beads is possible as shown in Examples 14-16 and 19 for otherPC-biotin conjugates.

Example 22 Isolation of Analytes Using Photocleavable Affinity CaptureAgents: Analyte Pre-Purification and Pre-Enrichment for Improved Signalto Noise Ratios in Downstream Assays

The goal will be to improve signal to noise ratios and eliminateinterference in downstream assays, such as traditional “sandwich”immunoassays, by pre-purifying and pre-enriching an analyte (e.g.antigen) using photocleavable antibodies. For example, as compared totraditional sandwich immunoassays (e.g. ELISA or microarray) whereanalyte pre-purification and pre-enrichment is not performed.

Preparation of a Photocleavable Antibody Affinity Matrix:

400 μg of Alexa Fluor® 488 conjugated rat anti-mouse IL-2 antibodypurchased from BD Biosciences (San Jose, Calif.; clone JES6-5H4 suppliedin 10 mM phosphate buffer 150 mM NaCl and 0.09% azide without proteincarrier; catalog number 557725) will be dialyzed, conjugated toPC-biotin, and pre-loaded to NeutrAvidin agarose beads in the samemanner as described in Example 2 for the anti-HSV antibody. The antibodywill be pre-loaded at saturating levels (5× molar excess) to ensuremaximum antibody density per unit volume of NeutrAvidin agarose beads.Note that according to the manufacturer's specifications, this antibodyis immunoprecipitation compatible as well as tested for detection insandwich ELISA assays. Additionally, the Alexa Fluor® 488 fluorescentlabel will be chosen due to its resistance to photo-bleaching. Themanufacturer supplied antibody solution is free of unlabeled antibodyand uncoupled fluorophore.

Antibody Microarray Printing:

A different and unlabeled rat anti-mouse IL-2 monoclonal antibody, cloneJES6-1A12, recognizing an epitope different from that of thephotocleavable IL-2 antibody prepared as described in the previousparagraph, will also be purchased from BD Biosciences (San Jose, Calif.;catalog 554424) and left untreated. The antibody will be left undilutedin its supplied phosphate buffer and printed to various microarraysurfaces using a GMS 417 robotic pin-and-ring microarraying instrument(Genetic Microsystems/AffyMetrix; Santa Clara, Calif.). As a negativecontrol, pre-immune non-specific rat IgG will be printed in equalamounts. Spots will be approximately 200 microns in diameter andapproximately 50 pL of applied volume each. Coated or activated glassmicroarray surfaces for printing will be amine-reactive aldehyde orepoxy activated substrates (SuperAldehyde or SuperEpoxy substrates,TeleChem International, Inc. ArrayIt™ Division, Sunnyvale, Calif.),amine derivatized substrates (GAPS II substrates, Corning IncorporatedLife Sciences, Acton, Mass.) and nitrocellulose coated substrates(SuperNitro Substrates, TeleChem International, Inc. ArrayIt™ Division,Sunnyvale, Calif.). Following printing, substrates will be washed 4×2min each with excess TBS-T and subsequently blocked for 30 min in 5% BSA(w/v) in TBS-T. Slides will then be rinsed 4× briefly (5 sec) inpurified water and dried.

Microarray Sandwich Immunoassay on Photocleavable Antibody Enriched andConcentrated Antigen:

As the test analyte (antigen), recombinant mouse IL-2 will be purchasedfrom R&D Systems (Minneapolis, Minn.; catalog number 402-ML-020/CF). TheIL-2 will then be exogenously added into normal mouse serum that isdevoid of detectable endogenous mouse IL-2 (i.e. validated sera fromnon-infected or compromised animals). The recombinant mouse IL-2 will besupplemented (diluted) into the serum from the high concentration stock(i.e. minimum 100× stock) to various final concentrations over thenormal range of sandwich immunoassay detection sensitivity (i.e. lowng/mL to pg/mL range) to determine the limits of sensitivity of themicroarray sandwich assay. The IL-2 will then be purified from thevarious supplemented sera using the photocleavable antibody affinitymatrix prepared as described earlier in this example (i.e. beadedaffinity matrix that is pre-loaded with a fluorescently labeledanti-mouse IL-2 photocleavable antibody). For purification, just enoughaffinity matrix will be added to the various supplemented sera toprovide a 2-fold molar excess binding capacity relative to the amount ofIL-2 present. At each IL-2 dilution tested, the total volume of IL-2supplemented serum added to the affinity matrix will be 100 μL, 1 mL or10 mL to ultimately yield concentrating factors of 1×, 10× and 100×respectively following photo-release (see later) of the isolated IL-2into 100 μL volume. Binding (capture) will be allowed to occur for 1 hrwith gentle mixing and the beaded affinity matrix will be washed 2×5 mineach then 2× briefly (briefly=5 sec vortex mix) with 50 bead volumes of0.1% BSA (w/v) in PBS. The fluorescently labeled antibody-antigencomplexes will then be photo-released from the beaded affinity matrixvia illumination of the bead suspension, with mixing, for 5 min withnear-UV light (365 nm peak UV lamp, Blak-Ray Lamp, Model XX-15, UVP,Upland, Calif.) at a 5 cm distance. Importantly, light illumination willbe performed directly in uncovered/uncapped polypropylenemicro-centrifuge tubes, such that there will be no solid barrier betweenthe bead suspension and the light source. The power output under theseconditions is 2.6 mW/cm at 360 nm, 1.0 mW/cm² at 310 nm and 0.16 mW/cm²at 250 nm. Photo-release will be performed into 100 μL of solution, justenough to overlay a standard sized microarray substrate (slide), andwill be performed with 0.1% BSA (w/v) in TBS as the buffer. Thephoto-released antibody-antigen complexes, now in solution (andseparated from beads), will then be applied to the antibody printedmicroarray substrate for recapture (thus forming theantibody-antigen-antibody “sandwich” on the array surface).Alternatively, the 100 μL suspension of beaded affinity matrix will bespread over the surface of the microarray substrate prior tophoto-release (e.g. using an overlaid glass coverslip that istransparent to near-UV) and photo-release will be performed by directlyexposing the overlaid microarray substrate to the light source.Recapture of the photo-released (fluorescent) antibody-antigen complexesonto the microarray-printed antibody will be allowed to occur for 1 hr.Unbound materials (and beads where applicable) will then be removed fromthe microarray substrate by 4× washes for 2 min each in TBS-T followedby 4× brief (5 sec) rinses in purified water. The microarray will bedried and the fluorescence signal read using an ArrayWoRx^(e) BioChipfluorescence reader (Applied Precision, LLC, Issaquah, Wash.) with theappropriate standard manufacturer supplied filter sets.

The anticipated results will be improvements in the signal to noiseratios and elimination of assay interference by pre-purifying andpre-enriching the analyte (the IL-2 antigen in this case) using aphotocleavable antibody prior to application to the microarray surface(note: photocleavable antibody serves dual purpose as detectionantibody). Comparisons will be made to the traditional sandwichimmunoassay format where analyte pre-purification and pre-enrichment isnot performed (e.g. crude analyte will be directly applied onto theantibody-printed microarray substrate, capture will be allowed to occur,the microarray will be washed and then treated with the fluorescentlylabeled detection antibody).

Example 23 Isolation of Protein Kinase C from Crude Cell Lysates UsingSecondary Photocleavable Antibodies Followed by Downstream KinaseActivity Assay Cell Activation:

Cultured HeLa cells (ATCC; Manassas, Va.) were stimulated for 5 min with200 nM Phorbol-12-Myristate-13-Acetate (PMA; EMD Biosciences, Inc., SanDiego, Calif.) and subsequently detergent fractionated into sub-cellularcompartments according to reported procedures [Ramsby et al. (1994)Electrophoresis 15, 265-277; Ramsby & Makowski. (1999) Methods Mol Biol112, 53-66; Chiang et al. (2000) J Biochem Biophys Methods 46, 53-68].PMA is a potent activator of PKCα and is well known to causetranslocation of the kinase from the cytosolic to the membranesub-cellular compartments [Ross & Joyner. (1997) Endothelium 5, 321-332;Bazan & Rapoport. (1996) JPharmacol Toxicol Methods 36, 87-95;Yazlovitskaya & Melnykovych. (1995) Cancer Lett 88, 179-183].

Kinase Isolation and Functional Assay:

A photocleavable antibody conjugated solid affinity matrix was preparedand used to isolate and photo-release the antigen into solutionessentially as described in Example 2 except that in this case, aphotocleavable anti-IgG secondary antibody was used to immobilize theunlabeled primary antibody onto the solid affinity matrix. This antibodyaffinity matrix was used to isolate and photo-release endogenous PKCafrom the undiluted detergent fractionated HeLa cell extracts. PKCccactivity, following photocleavable antibody mediated purification, wasassayed using a non-isotopic heterogeneous ELISA-type kit available fromEMD Biosciences, Inc. (San Diego, Calif.) consisting of an immobilizedpeptide substrate and an anti-phospho-peptide antibody mediateddetection system (calorimetric signal generation).

Results:

The goal is to improve signal to noise ratios and eliminate potentialinterference from contaminants or similar kinases (e.g. other PKCisoforms specific for the same substrates, such as PKCβ by employing apre-purification step based on photocleavable antibodies. The results inFIG. 20 demonstrate that, based on functional activity measurements ofphotocleavable antibody isolated HeLa cell PKCα, translocation of thekinase from the cytosol to the membrane compartments was clearlyobserved in correlation with the scientific literature [Ross & Joyner.(1997) Endothelium 5, 321-332; Bazan & Rapoport. (1996) J PharmacolToxicol Methods 36, 87-95; Yazlovitskaya & Melnykovych. (1995) CancerLett 88, 179-183]. FIG. 20 shows a baseline PKCα distribution of 79±7%cytosol and 21±2% membrane which shifts to 16±4% cytosol and 84±3%membrane following PMA stimulation of the cultured HeLa cells (t-test pvalue of 0.000003; n=4) (distribution confirmed by Western blot).

Example 24 Contact Photo-Transfer from Individually Resolved Beads in aThin Liquid Film Under a Cover Glass Using a PC-Antibody Preparation ofa Photocleavable Antibody Affinity Matrix:

The photocleavable antibody beaded affinity matrix was prepared usingthe monoclonal anti-HSV tag antibody (EMD Biosciences, Inc., San Diego,Calif.) as described in Example 2.

Cell-Free Expression and tRNA Mediated Labeling:

Human glutathione-s-transferase (GST) and the p53 oncoprotein, bothcontaining an HSV epitope tag on the C-terminus, were expressed in acell-free reaction as described earlier in Example 1 with the followingexceptions: Only AmberGen's BODIPY-FL-tRNA^(COMPLETE) was used at 2 μMfor labeling and not the PC-biotin-tRNA^(COMPLETE) or any othermisaminoacylated tRNA labeling reagents. The 2 different DNA species,for GST and p53, were mixed at a 1:1 ratio and co-expressed in the samereaction. The expression reaction size was only 50 μL instead of 200 μL.Importantly, the aforementioned anti-HSV tag photocleavable antibodyaffinity beads were added directly into the expression reaction, at thestart of the expression reaction, as the last component. To do this, 5μL of the beads was washed 3×400 μL briefly (briefly 5 sec vortex mix)in TBS using 0.45 micron pore size, PVDF membrane, micro-centrifugeFiltration Devices to facilitate manipulation of the small volume ofaffinity matrix (˜100 micron beads) and exchange the buffer(Ultrafree-MC Durapore Micro-centrifuge Filtration Devices, 400 μLcapacity; Millipore, Billerica, Mass.). The expression reaction mixturewas used to resuspend the washed bead pellet and the bead suspensiontransferred to a fresh 0.5 mL polypropylene tube. The expressionreaction was carried out in the presence of the beads for 45 min at 30°C. The TDB buffer used to stop the expression reaction and prepare thesample contained no BSA or any other protein carrier and additionallycontained 4 mM cycloheximide. After addition of the TDB buffer, thesamples were immediately processed for washing and isolation.

Isolation of Labeled Nascent Proteins:

The washing and isolation procedure was performed in batch mode using0.45 micron pore size, PVDF membrane, micro-centrifuge FiltrationDevices to facilitate manipulation of the small volumes of affinitymatrix (˜100 micron beads) and exchange the buffers (Ultrafree-MCDurapore Micro-centrifuge Filtration Devices, 400 μL capacity;Millipore, Billerica, Mass.). All steps were performed at +4° C. or onan ice water bath and all reagents and samples were also kept underthese conditions during the procedure. After cell-free expression in thepresence of the anti-HSV tag photocleavable antibody affinity beads,beads were washed by mixing 2× briefly (briefly=5 sec vortex mix) and 1×for 5 min in 400 bead volumes per wash. The buffer used for washing thebeads was PBS pH 7.5 and 5 mM DTT. The beads were then additionallywashed 1× briefly (briefly=5 sec vortex mix) in 400 bead volumes of 50%glycerol and 5 mM DTT in PBS. Prior to contact photo-transfer of thecaptured and isolated proteins, the washed pellet of 1 μL of beads wassuspended in a final volume of 200 μL with 50% glycerol and 5 mM DTT inPBS thereby resulting in a 0.5% (v/v) bead suspension that can be storedlong-term at −20° C. without freezing of the sample and thus withoutdamage to the agarose beads.

Contact Photo-Transfer from Individually Resolved Beads:

The 0.5% bead suspension containing the captured proteins wasresuspended by vortex mixing and 10 μL of suspension was manuallypipetted to the surface of epoxy activated glass microarray substrates(slides) (SuperEpoxy substrates, TeleChem International, Inc. ArrayIt™Division, Sunnyvale, Calif.). The 10 μL pool containing the beads wasthen overlaid with a standard circular 12 mm microscope cover glass,creating a thin film of fluid between the cover glass and the microarraysubstrate. The microarray substrate overlaid with the cover glass wasallowed to stand for 5 min without disturbance. The substrates were thenilluminated, through the cover glass, without agitation or disturbance,for 5 min with near-UV light (365 nm peak UV lamp, Blak-Ray Lamp, ModelXX-15, UVP, Upland, Calif.) at a 5 cm distance to photo-release andtransfer the target proteins. The power output of the lamp under theseconditions was 2.6 mW/cm² at 360 nm, 1.0 mW/cm² at 310 nm and 0.16mW/cm² at 250 nm. After light treatment, the microarray substrateoverlaid with the cover glass was incubated without disturbance for 30min at 37° C. in a sealed and humidified chamber to fully ensurephoto-released proteins react with the activated solid surface. Thebeads and any unbound protein as well as the overlaid cover glass wasthen removed with 3× brief (5 sec) washes in TBS-T followed by 4× brief(5 seq) washes in purified water. Phase contrast light microscopyreveals that the easily visible 100 micron agarose beads were completelywashed/removed from the glass substrates. The slides were dried prior tofluorescence imaging.

Detection of Photo-Transferred Protein:

Detection of the directly incorporated tRNA mediated BODIPY-FLfluorescence labeling was achieved by imaging the dry microarraysubstrates on an ArrayWoRx^(e) BioChip fluorescence reader (AppliedPrecision, LLC, Issaquah, Wash.) using the appropriate manufacturersupplied standard filter set and the resolution set to 9.7 microns.

Results:

Results are shown in FIG. 21. The BODIPY-FL fluorescence image showsmultiple sharply resolved and non-clustered spots corresponding to thelabeled protein material that was contact photo-transferred from theaffinity beads. The spots average roughly 100 μm in diameter,correlating with the approximate diameter of the beads.

Example 25 Cell-Free Protein Synthesis and In Situ ProteinImmobilization on Beads Followed by Contact Photo-Transfer UsingPhotocleavable Antibodies Immobilization of Expression DNA on Beads:

Genes encoding human glutathione-s-transferase (GST) and the p53oncoprotein were used in this example. Cloned and purified expressionplasmids described in Example 1 and containing the aforementioned geneinserts were used as the template for PCR amplification with universalprimers. Forward and reverse primers were directed against commonsequences in the expression plasmid such that the PCR ampliconscontained the elements needed for efficient cell-free expression (T7 RNApolymerase promoter and ribosome binding site), the gene insert, as wellas the common C-terminal polyhistidine tag and HSV epitope tag. The PCRprimers were custom purchased commercially from Sigma-Genosys (TheWoodlands, Tex.) and importantly, the reverse primer contained a 5′biotin modification for immobilization of the PCR amplicons. PCR primersequences were as follows:

[SEQ NO. 1] Forward: 5′CgTCCCgCgAAATTAATACgACTCAC3′ [SEQ NO. 2] Reverse:5′[Biotin]gTTAAATTgCTAACgCAgTCAggAg3′

PCR was performed using standard practices and a commercially availablekit according to the manufacturer's instructions (SuperTaq™ DNAPolymerase Kit; Ambion, Austin, Tex.). The following thermocycling stepswere used for the PCR reaction: Initially 94° C. 2 min (once) and then25 cycles of 94° C. 30 s, 55° C. 30 s and 72° C. 30 s to 2 min(depending on DNA length), followed by a final 72° C. 10 min (once).Purification and concentration of the PCR amplicons was achieved using acommercially available kit according to the manufacturer's instructions(QIAquick PCR Purification Kit; Qiagen, Valencia, Calif.). Resultantpurified DNA concentrations ranged from 0.15 to 0.2 μg/μL. The correctsize and integrity of the PCR amplified DNA was verified by standardagarose gel electrophoresis and ethidium bromide staining withcomparison to a known molecular weight ladder (molecular weightstandards). Single sharply resolved bands were observed for each PCRamplicon at the correct molecular weight positions without anydetectable contaminants or degradation products. Expression of thesoluble PCR amplicons was validated using the rabbit reticulocytecell-free expression system described in Example 1 coupled withselective labeling with AmberGen's BODIPY-FL-tRNA^(COMPLETE) andfollowed by SDS-PAGE and fluorescence imaging as described in earlierExamples. Expression efficiency of the PCR amplicons was found to becomparable to the starting plasmid DNA template.

Next, to immobilize the biotin-DNA on beads, 10 μL of streptavidinconjugated 4% cross-linked agarose beads (˜100 microns diameter;Sigma-Aldrich; St. Louis, Mo.) were first washed 3×400 μL in TE-NaClbuffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 2M NaCl) usingmicro-centrifuge Filtration Devices according to the manufacturer'sinstructions (0.45 micron pore size, PVDF membrane, Ultrafree-MCDurapore Micro-centrifuge Filtration Devices, 400 μL capacity;Millipore, Billerica, Mass.). The washed bead pellets were resuspendedwith 150 μL of the purified PCR amplified biotin-DNA diluted to 10 ng/μLin TE-NaCl buffer (1.5 μg total DNA). The DNA was allowed to bind for 30min with gentle mixing. Again using the micro-centrifuge FiltrationDevices, the beads were washed 3×400 μL with TE-NaCl buffer followed by1×400 μL in 50% glycerol/50% TE-NaCl buffer. The beads were then dilutedto a 10% suspension (v/v) with the 50% glycerol/50% TE-NaCl buffer andstored at −20° C. The amount of DNA captured was calculated to be 27%(0.04 μg per μL beads) by comparing the absorbance at 260 nm of thestarting DNA solution to the DNA solution after incubation with thebeads. It is important to note that this level of DNA capture on thestreptavidin beads was well below the saturation limit of thestreptavidin beads according to the manufacturer's specifications (˜30ng free d-biotin or roughly 2 μg of biotinylated macro-molecule, such asan antibody, per μL of bead volume). Thus, sufficient biotin bindingcapacity was expected to remain for capture of photocleavable biotin(PC-biotin) labeled antibodies as described later in this Example.

For qualitative verification of DNA binding to the streptavidin agarosebeads, the beads were stained with PicoGreen (Invitrogen Corporation,Carlsbad, Calif.). The PicoGreen reagent binds selectively todouble-stranded DNA and upon binding undergoes a roughly 1,000 foldfluorescence enhancement. 5 μL bead volume of the prepared beads waswashed 3×400 μL with TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) using themicro-centrifuge Filtration Devices. The beads were resuspended in 50 μLof PicoGreen reagent diluted 1/200 in TE buffer and the suspensiontransferred to 0.5 mL clear, thin-walled, polypropylene PCR tubes. Thebeads were briefly centrifuged to form a pellet and without removing thefluid, the bead pellets were scanned for fluorescence directly in thetubes using a FluorImager SI laser-based scanner (MolecularDynamics/Amersham Biosciences Corp., Piscataway, N.J.). As a negativecontrol, plain streptavidin beads without bound DNA were also stainedwith PicoGreen as a blank. Results shown in FIG. 22A. The fluorescencesignal coming from the bead-bound DNA is clearly visible from the beadpellet with an integrated signal intensity of 40:1 relative to the plainstreptavidin beads (blank beads) without any bound DNA.

Immobilization of PC-Antibody on DNA Encoded Beads:

To generate the photocleavable antibody (PC-antibody), the mousemonoclonal anti-HSV tag antibody (EMD Biosciences, Inc., San Diego,Calif.) directed against the common HSV epitope tag present in allexpressed proteins was labeled with photocleavable biotin (PC-biotin).To perform labeling, the antibody was left in the manufacturer suppliedbuffer (1 μg/μL antibody, 50% glycerol, PBS, 0.02% sodium azide) andsupplemented with 1/9 volume of 1M sodium bicarbonate to yield a finalsodium bicarbonate concentration of 100 mM. 330 μg of the antibody (nowin 367 μL) was then labeled using 20 molar equivalents of AmberGen'sPC-biotin-NHS reagent (added from 50 mM stock in DMF) for 30 min withgentle mixing and protected from light. Un-reacted and hydrolyzedPC-biotin-NHS reagent was removed by running the antibody through aNAP-10 Sepharose G-25 desalting column (Amersham Biosciences Corp.,Piscataway, N.J.) according to the manufacturer's instructions exceptthat only the first 1 mL of elution was collected and used. For thecolumn, TBS was used as the equilibration and elution buffer. Theconcentration of the resultant antibody was measured by absorbance at280 nm to be 0.21 μg/μL. The antibody was separated into aliquots andstored frozen at −70° C.

Next, the prepared PC-antibody was co-immobilized onto the prepared DNAencoded streptavidin beads described earlier in this Example. To do so,5 μL bead volume of the DNA encoded beads was washed 3×400 μL withTE-Saline (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 200 mM NaCl) using themicro-centrifuge Filtration Devices. The beads were then transferred tolow protein binding 0.5 mL polypropylene PCR tubes (Eppendorf NorthAmerica, Westbury, N.Y.) and all fluid supernatant was removed leavingonly the hydrated bead pellet. The stored PC-antibody preparation at0.21 μg/mL in TBS, described earlier in this example, was diluted to0.14 μg/μL with TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) andsupplemented to 1 mM EDTA final concentration from a 500 mM, pH 8.0 EDTAstock solution. 150 μL of the diluted PC-antibody solution was used toresuspend the washed DNA encoded bead pellet and the suspension wassubsequently mixed gently for 15 min protected from light. Using themicro-centrifuge Filtration Devices, the beads were then washed 3×400 μLwith TE-Saline buffer followed by 1×400 μL with 50% glycerol/50% TEbuffer/200 mM NaCl. Beads were then resuspended to a 10% (v/v)suspension in 50% glycerol/50% TE buffer/200 mM NaCl and stored at −20°C. The binding of the PC-antibody to the beads was monitored byabsorbance at 280 nm of the starting diluted antibody solution versusthe antibody solution after incubation with the beads. A calculated 26%of the added antibody was captured for a loading of approximately 1 μgof PC-antibody per μL of bead volume.

Expression and In Situ Immobilization of Proteins with DNA EncodedPhotocleavable Antibody Beads:

Briefly, the mechanism for multiplexed cell-free protein expression within situ protein capture involves affinity capture of proteins on asurface, simultaneously as they are cell-free produced using thesurface-immobilized DNA as a template (DNA co-immobilized with affinitycapture agent), with capture occurring locally at the position of theparent immobilized DNA.

For cell-free protein expression and in situ immobilization, the beadsprepared with co-immobilized expression DNA and photocleavable (PC)anti-HSV antibody were used. Preparation of such beads was describedearlier in this Example. Beads encoded with GST DNA were used for thisparticular demonstration. Just prior to the cell-free expressionreaction, 1 μL of beads (i.e. 1 μL bead volume; roughly 750 beads) wasadditionally washed (in addition to washes done in their preparation)1×400 μL with nuclease free water using the micro-centrifuge FiltrationDevices. The rabbit reticulocyte cell-free expression reaction mixturewas prepared as described in Example 1 except that only 1μBODIPY-FL-tRNA^(COMPLETE) was used for labeling the nascent proteinsand in one case, soluble expression DNA was not added, but instead wasreplaced with the 1 μL of GST DNA encoded PC-antibody beads. To add thebeads into the cell-free reaction mixture, the reaction mixture was usedto resuspend the washed 1 μL bead pellet. 50 μL of expression reactionmixture was used for each 1 μL bead pellet. As a negative control, asecond expression reaction received plain streptavidin beads whichlacked both the bound GST DNA and bound PC-antibody, but the reactionsample was supplemented with validated soluble plasmid DNA (forexpressing GST) as described in Example 1. A third expression reactionreceived the 1 μL of GST DNA encoded PC-antibody beads but was alsoadditionally supplemented with validated soluble plasmid DNA (forexpressing GST).

The expression reaction was carried out for 1 hr at 30° C. with gentlemixing. The reaction was then mixed with equal volume of TranslationDilution Buffer (TDB) as described in Example 1 except that the buffercontained 10 mM DTT instead of 2 mM and additionally contained 20 mMEDTA added from a 500 mM pH 8.0 stock and 4 mM cycloheximide(Sigma-Aldrich, St. Louis, Mo.) added from a 355 mM stock in DMSO. TheTDB contained no BSA or other protein carriers. The samples wereequilibrated in the buffer for 15 min at +4° C. with gentle mixing.Using the micro-centrifuge Filtration Devices, the beads were washed3×400 μL with PBS containing 5 mM DTT. The beads were then washed 1×400μL with 50% glycerol, PBS and 5 mM DTT and diluted with the same bufferto a 0.5% (v/v) bead suspension.

Contact Photo-Transfer from Individually Resolved Beads:

Contact photo-transfer from individually resolved beads onto epoxyactivated glass microarray substrates (slides) (SuperEpoxy substrates,TeleChem International, Inc. ArrayIt™ Division, Sunnyvale, Calif.)overlaid with a cover glass was performed as described in Example 24.

Detection of Photo-Transferred Protein.

Detection of the directly incorporated tRNA mediated BODIPY-FLfluorescence labeling was achieved by imaging the dry microarraysubstrates on an ArrayWoRx^(e) BioChip fluorescence reader (AppliedPrecision, LLC, Issaquah, Wash.).

Results:

Results are shown in FIG. 22B. The top center panel shows thefluorescence image following contact photo-transfer from beads thatlacked both immobilized GST DNA and PC-antibody, but where validatedsoluble plasmid DNA was included in the expression reaction tofacilitate GST protein production. Note that the expressed GST does notbind the beads and is therefore not transferred to the microarraysubstrate. The lower left panel shows contact photo-transfer from beadsthat contained both immobilized DNA and immobilized PC-antibody againstthe C-terminal HSV epitope tag present in expressed GST. GST isexpressed from the immobilized DNA and labeled with theBODIPY-FL-tRNA^(COMPLETE). The nascent GST is bound by thebead-immobilized PC-antibody and is subsequently contactphoto-transferred from individual beads to the microarray substrate,leaving ˜100 micron diameter fluorescent microarray features. OtherExamples and experiments verify that the fluorescence in fact comes fromthe labeled nascent protein and not the BODIPY-FL-tRNA^(COMPLETE). Thelower right panel shows contact photo-transfer from beads that containedboth immobilized DNA and immobilized PC-antibody against the C-terminalHSV epitope tag present in expressed GST and where the expressionreaction was additionally supplemented with soluble validated plasmidDNA. This permutation shows significantly increased signal due to theadded soluble expression DNA and shows that the immobilized DNA alone(lower left panel) does not produce enough protein to saturate thebead-bound PC-antibody.

Example 26 Cell-Free Protein Synthesis and In Situ Protein Imobilizationon Beads Followed by Contact Photo-Transfer Using PhotocleavableAntibodies or tRNA Mediated Labels: Co-Expression of Mixed DNA EncodedBead Species in a Single Reaction Immobilization of Expression DNA onBeads:

This Example is similar to Example 25 except that different bead speciesbearing DNA for expression of different proteins were translated in thesame cell-free reaction for multiplexed protein production and in situprotein capture on the parent DNA encoded beads. Additionally, examplesare described using either PC-antibodies or tRNA mediated photocleavablelabels for in situ protein capture on the parent DNA encoded beads.

Genes encoding human glutathione-s-transferase (GST) and the p53oncoprotein were amplified by PCR with a biotin modified primer and theamplicons attached to streptavidin agarose beads as described in Example25.

Immobilization of PC-Antibody on DNA Encoded Beads:

To generate the photocleavable antibody (PC-antibody), the mousemonoclonal anti-HSV tag antibody (EMD Biosciences, Inc., San Diego,Calif.) directed against the common HSV epitope tag present in allexpressed proteins was labeled with photocleavable biotin (PC-biotin)and co-immobilized on the aforementioned DNA encoded streptavidin beadsas described in Example 25.

Expression and In Situ Immobilization of Proteins with DNA EncodedPhotocleavable Antibody Beads:

For cell-free protein expression and in situ immobilization, the beadsprepared with co-immobilized expression DNA and photocleavable (PC)anti-HSV antibody were used. Preparation of such beads was describedearlier in this Example. Beads encoded with GST DNA or p53 DNA were usedfor this particular demonstration. Cell-free expression in the presenceof the beads was performed essentially as described in Example 25 withthe following exceptions: Only the bead-immobilized DNA was used forexpression as no soluble DNA was added in any case. To ensure the DNAwas tightly bound to the beads, the GST and p⁵³ DNA encoded beads wereadditionally washed separately (in addition to washes performed in theirpreparation). Approximately 1 μL bead volume was washed 1×400 μL inTE-Saline (see Example 25) at 30° C. for 30 min with gentle mixing.Beads were then separated from the fluid wash using the micro-centrifugeFiltration Devices (see Example 25; all subsequent bead washings use theFiltration Devices) and washed 1× briefly (briefly=5 sec vortex mix)with 400 μL of TE-Saline. Beads were then resuspended to 300 μL withTE-Saline and the appropriate amount of bead suspension was combined toyield a 1:1 bead mixture containing 0.5 μL bead volume of GST DNAencoded beads and 0.5 μL bead volume of p53 DNA encoded beads. The fluidwas removed from the bead mixture using the Filtration Device and thebeads washed 1×400 μL with nuclease-free water just prior to combiningwith the cell-free expression reaction mixture as described in Example25.

The cell-free expression reaction in the presence of the beads wasperformed as described in Example 25 except that the reaction wasperformed for 15 min at 30° C. without mixing or shaking and after thereaction, the samples were not mixed with TDB buffer but wereimmediately washed. For washing, the expression reaction was immediatelytransferred to new Filtration Devices and the fluid removed from thebeads by filtration (all subsequent bead washes performed in FiltrationDevices). Beads were then washed 1×350 μL briefly (briefly=5 sec vortexmix) with ice cold PBS containing 5 mM DTT. The beads were then washed4×400 μL briefly (briefly=5 sec vortex mix) with the same ice coldbuffer. Lastly, beads were washed 1×400 μL briefly (briefly=5 sec vortexmix) with ice cold 50% glycerol, PBS and 5 mM DTT and diluted with thesame buffer to a 0.5% (v/v) bead suspension.

Contact Photo-Transfer from Individually Resolved Beads:

Performed as described in Example 25 except that after contactphoto-transfer, washing and drying of the microarray slides, the slideswere further processed for antibody probing as described in thefollowing paragraphs.

Preparation of an Anti-p53-Cy5 Fluorescent Antibody:

While the BODIPY-FL-tRNA^(COMPLETE) provides green fluorescence labelingof all nascent cell-free expressed proteins, a protein specific antibodywas needed to distinguish between the different proteins (GST and p53)that were contact photo-transferred from the different DNA encodedPC-antibody beads. For this, an anti-p53 monoclonal antibody wasconjugated to the Cy5 fluorescent dye to be used in probing themicroarray substrate containing the contact photo-transferred proteinspots. For this mouse monoclonal anti-p53 clone BP53-12 was purchasedfrom Biosource International (Camarillo, Calif.). The antibody issupplied purified at 1 μg/μL (100 μL for 100 μg) in PBS buffer only. Theantibody is then supplemented with 1/9 volume of 1M sodium bicarbonateto give a 100 mM final concentration of sodium bicarbonate. The antibodywas then labeled by adding the Cy5—NHS monoreactive ester (AmershamBiosciences Corp., Piscataway, N.J.) from a 27 mM stock (stock in DMSO).The Cy5—NHS ester was added to a 12-fold molar excess relative to theantibody. The labeling reaction was allowed to proceed by gentle mixingfor 30 min protected from light with aluminum foil. Unreacted/hydrolyzedlabeling reagent was removed from the labeled p53 antibody using aMicroSpin G-25 desalting column (Amersham Biosciences Corp., Piscataway,N.J.) according to the manufacturers instructions (except that thecolumn was additionally pre-washed 1×350 μL with TBS). 2 columns wereused with a loading of approximately 55 μL per column and the elutedantibody pooled afterwards. The Cy5 labeled and purified p53 antibodywas measured in a spectrophotometer for absorbance at 280 nm (proteinconcentration) and 649 mm (Cy5 concentration). Using the appropriateextinction coefficients, the result was 0.56 μg/μL antibodyconcentration and a calculated average of 2.8 Cy5 molecules per moleculeof antibody.

Probing the Microarray with Anti-p53-Cy5 Antibody:

The aforementioned anti-p53-Cy5 antibody was used to probe themicroarray slide. To do so, the slides were first blocked for 30 min at37° C. with 5% BSA (w/v) in TBS-T. Next, the slide was probed with ˜15mL of the anti-p53-Cy5 antibody diluted 1/1,000 (0.56 μg/mL) with 5% BSA(w/v) in TBS-T. Probing was performed with mixing in a tray for 30 minat 37° C. The microarray slides were then washed for 4×2 min each withexcess TBS-T (cover glass removed on first wash), followed by 4× briefly(5 sec) with purified water and dried prior to imaging as describedlater. Separately, using this same procedure, the anti-p53-Cy5 antibodyconjugate was validated to selectively stain the cell-free expressed p53protein and without any detectable cross-reactivity with cell-freeexpressed GST.

Detection of Photo-Transferred Protein:

Detection of the directly incorporated tRNA mediated BODIPY-FLfluorescence labeling as well as signal from the Cy5 labeled anti-p53antibody was achieved by imaging the dry microarray substrates on anArrayWoRx^(e) BioChip fluorescence reader (Applied Precision, LLC,Issaquah, Wash.) using the appropriate standard manufacturer suppliedfilter sets.

Results:

Fluorescence microarray images are shown in FIG. 23A. The top panel is agray-scale image from the green fluorescence channel corresponding tothe internal BODIPY-FL tRNA mediated protein labeling. The bottom panelis a gray-scale image of the same slide and same area from the redfluorescence channel corresponding to the binding of the anti-p53-Cy5antibody to the protein spots on the microarray. Quantification of theintegrated intensities for each spot in both the red and greenfluorescence images was performed and the ratio of red to greenfluorescence is shown for each spot in FIG. 23B. In this Example,partial protein cross-over is observed, i.e. escape of nascent p53 fromthe parent p53 DNA encoded bead and cross-contamination (capture byPC-antibody) onto the GST DNA encoded beads, and presumably the converseas well. Nonetheless, 2 distinct species of spots originating from thebeads (38 beads total analyzed) are observed. The 2 species of spots areidentified by differing red fluorescence (p53 content) to greenfluorescence (total nascent protein content) ratios as shown in FIG.23B. The spots arising from p53 DNA encoded beads have a red:green ratioof 1.13±0.23 (visible as yellow spots in the color image overlay FIG.23A bottom panel) while the spots arising from GST DNA encoded beadshave a ratio of 0.30±0.12 (visible as green spots in the color imageoverlay FIG. 23A bottom panel). The 2 populations of beads werestatistically analyzed using an unpaired 2-tailed t-test and determinedto be very significantly different with a p value of 0.000000000002 (pvalue<0.05 considered significant with 95% confidence). Furthermore, thenumber of each species of spots is at an approximate a 1:1 ratio (17spots p53 to 21 spots GST) as expected from the 1:1 mixing of the 2 beadspecies.

As shown earlier, there is some occurrence of nascent proteins escapingfrom their parent DNA encoded bead resulting in partialcross-contamination of other non-parent beads. This problem arises frommixing and diffusion rates that occur in the 3-dimensional bulk fluidexpression reaction as well as from settling of the beads, by gravity,to the bottom of the reaction tube and into very close proximity to eachother. This problem can be solved by modulating parameters including theDNA to PC-antibody ratio on the beads as well as the expression reactiontimes and temperature and the ratio of beads to expression mixture.Additionally, specialized techniques can be used to solve this problem,such as the inclusion of soluble (i.e. not tethered to beads) epitopetag antibody (i.e. anti-HSV antibody in this case) into the expressionreaction to bind-up any nascent proteins that escape their micro-porousparent bead matrix (i.e. not captured by the tethered PC-antibody). Amore advanced method uses the soluble epitope tag antibodyconjugated/attached to a large soluble polar macromolecule (e.g. largedextrans or large irrelevant non-expression DNA plasmids) such that theantibody fails to enter the micro-pores of the cross-linked agarosebeads (by size/charge exclusion). With this approach, the soluble freeantibody conjugate binds-up only proteins that escape the micro-porousbeads but does not interfere with PC-antibody mediated in situ proteincapture occurring within the micro-environment (porous matrix) of thebeads. This unique design is only effective with micro-porous beads suchas cross-linked agarose, and not with non-porous beads such as thestreptavidin conjugated 1 micron diameter magnetic beads from DynalBiotech (Dynabeads® MyOne™ Streptavidin; Dynal Biotech LLC, Brown Deer,Wis.) containing only an external monolayer of streptavidin. Therefore,if these non-porous 1 micron beads are loaded with DNA and PC-antibodyfor multiplexed expression and in situ protein capture, the cell-freeexpression reaction can be supplemented with an excess of larger (˜100micron diameter), porous, cross-linked agarose beads (e.g. 4% agarosebeads with 30 nm average pore size from Sigma-Aldrich, St. Louis, Mo.)bearing only tethered and non-cleavable epitope tag antibody (i.e.anti-HSV antibody in this case). These larger cross-linked agarose beadsbind-up only proteins that are not in situ captured onto the parent DNAencoded 1 micron magnetic beads, thus preventing beadcross-contamination. Since virtually all of the binding capacity of thelarger agarose beads is internal to the cross-linked beaded matrix, thetethered antibody will not interact with proteins that do not escape thesurface of the parent DNA encoded 1 micron magnetic beads. After theexpression reaction, the larger agarose beads can be separated from thesmaller magnetic beads by applying a magnet or by simple mesh filtering.An alternative strategy for preventing bead cross-contamination involvesexpression from the DNA encoded PC-antibody beads in a thin film offluid (the liquid cell-free expression reaction) containing the beads,such as is created by trapping (“sandwiching”) the fluid-bead suspensionbetween a standard glass microscope slide which is overlaid with astandard microscope cover glass. This design disperses the beads asdemonstrated in Example 24, which minimizes the possibility ofcross-contamination. This design also restricts protein diffusion andhence restricts nascent protein escape from the parent DNA encoded beadand subsequent cross-contamination of non-parent beads.

A variant of the overall bead-based multiplexed protein expression andcontact photo-transfer method presented in this Example involves in situnascent protein capture onto the parent DNA encoded bead [bead alsocontaining bound (strept)avidin with available biotin binding sites] viaa directly incorporated PC-biotin label by using AmberGen'sPC-biotin-tRNA^(COMPLETE). This variant does not use PC-antibodies (e.g.no PC-antibody to the HSV epitope tag is used) and therefore does notrequire genetically engineered epitope tags in the expressed proteins.The PC-biotin-tRNA^(COMPLETE) is not pre-bound to the bead surface butinstead is included in the solution-phase of the bead containingcell-free expression reaction. Once the expression reaction isinitiated, it sets off 2 competing processes, whereby a fraction of thePC-biotin-tRNA^(COMPLETE) is captured on the beads prior toparticipating in the translation reaction while another fraction of thetRNA is first utilized in the translation reaction to label the nascentprotein followed by in situ capture of the labeled nascent protein ontothe DNA encoded parent bead. As before, this relies on the ability ofthe immobilized expression DNA to localize the translation reaction tothe parent bead. When this process is performed as otherwise describedearlier in this Example (mixed beads in a single expression reaction),in conjunction with the contact photo-transfer method, similar proteinsegregation onto the parent DNA encoded beads and hence in thephoto-transferred spots is observed.

Example 27 Contact Photo-Transfer from 10 Micron Diameter Polymer Beadsfor High Density Arrays Preparing NeutrAvidin Coated Beads:

The beads used are 10.2±0.09 microns in diameter and composed of ahydrophobic styrene-divinylbenzene co-polymer and are commerciallyavailable from Bangs Laboratories, Inc. (Fishers, Ind.). The beads arecoated with the biotin binding protein NeutrAvidin (PierceBiotechnology, Inc., Rockford, Ill.) by passive adsorption. A second setof beads is coated with BSA as a negative control. To do so, 57 mg ofbeads was washed 3×400 μL each with 20 mM sodium phosphate, pH 6.3 and150 mM NaCl. Washes were performed by ˜5 sec vortex mixing. To wash thebeads or exchange the buffers, 0.45 micron pore size, PVDF membrane,micro-centrifuge Filtration Devices were used unless otherwise noted(Ultrafree-MC Durapore Micro-centrifuge Filtration Devices, 400 μLcapacity; Millipore, Billerica, Mass.). After washing, the beads wereresuspended in 400 μL of NeutrAvidin or BSA at a 2.5 mg/mL concentrationin 20 mM sodium phosphate, pH 6.3 and 150 mM NaCl. Binding was allowedto occur for 2 hr at 37° C. with gentle mixing. Beads were then washedfor 4×400 μL with 5% BSA (w/v) in TBS. Washes were performed by ˜5 secvortex mixing. Beads were then blocked for 15 min at 37° C. in the samebuffer. The beads were then washed for 3×400 μL with 0.1% sodium azideas a preservative in TBS. Washes were performed by ˜5 sec vortex mixing.Beads were resuspended to a 10% (v/v) suspension in the same buffer andstored at +4° C.

Conjugating Photocleavable Biotin & Cy5 to the Casein Test Protein:

Bovine casein (sodium salt; Sigma-Aldrich, St. Louis, Mo.) is labeledwith both photocleavable biotin (PC-biotin) and the fluorophore Cy5. Todo so, the casein was prepared to 2 mg/mL in 200 mM sodium bicarbonateand 200 mM NaCl. Any un-dissolved particulate was removed by passing thesolution through 0.45 micron pore size, PVDF membrane, micro-centrifugeFiltration Devices (Ultrafree-MC Durapore Micro-centrifuge FiltrationDevices, 400 μL capacity; Millipore, Billerica, Mass.). The filtrate wasthen collected and desalted on a NAP-10 Sepharose G-25 column (AmershamBiosciences Corp., Piscataway, N.J.) against the same 200 mM sodiumbicarbonate and 200 mM NaCl buffer according to the manufacturer'sinstructions. The protein concentration was then determined by measuringthe absorbance at 280 nm on a spectrophotometer (0.84 absorbance unitsin 1 cm path cuvette=1 mg/mL). The resultant recovered casein (1.2 mg/mLat 1 mL) was labeled using 10 molar equivalents of AmberGen'sPC-biotin-NHS reagent (added from 50 mM stock in DMF) for 20 min withmixing. Next, the casein was additionally labeled with the Cy5fluorophore using a Cy5—NHS monoreactive ester (Amersham BiosciencesCorp., Piscataway, N.J.) labeling reagent; The Cy5 labeling reagent wasadded to a 2.7-fold molar excess relative to the PC-biotin labeledcasein from a 27 mM stock prepared in DMSO. The reaction was allowed toproceed for 30 min with gentle mixing and protected from light. Thelabeled casein was then purified to remove any un-reacted or hydrolyzedlabeling reagent by using a NAP-10 Sepharose G-25 column (AmershamBiosciences Corp., Piscataway, N.J.) against a TBS buffer according tothe manufacturer's instructions. The labeled casein was stored at +4° C.protected from light.

Loading the PC-Biotin-Casein-Cy5 Conjugate to the NeutrAvidin Coated 10Micron Beads:

Both the NeutrAvidin coated and negative control BSA coated 10 microndiameter beads are treated with the PC-biotin-casein-Cy5 conjugate toallow binding to occur, which is expected only in the case of theNeutrAvidin beads. To do so, 100 mL of the 10% (v/v) coated bead stockswas mixed with 100 mL of 0.1 μg/μL of the PC-biotin-casein-Cy5 conjugatediluted in 5% BSA (w/v) in TBS. Binding was allowed to occur for 30 minwith gentle mixing. Using the aforementioned Filtration Devices, beadswere then washed 1×400 mL with 5% BSA (w/v) in TBS, 4×400 μL with PBSand 1×400 μL with 50% glycerol (v/v) and 5 mM DTT in PBS. All washeswere for 5 see vortex mixing followed by filtration. The beads wereresuspended to a 2% (v/v) suspension with 50% glycerol (v/v) and 5 mMDTT in PBS.

Contact Photo-Transfer from the PC-Biotin-Casein-Cy5 loaded 10 MicronBeads:

For contact photo-transfer, 0.5 mL of the prepared 2% (v/v) beadsuspensions were deposited onto the surface of epoxy activated 25×75 mmrectangular microarray substrates (SuperEpoxy substrates, TeleChemInternational, Inc. ArrayIt™ Division, Sunnyvale, Calif.). The 0.5 μLdroplets on the microarray substrates were then each overlaid with 12 mmdiameter round microscope cover glasses, which were then pressed gently.This limiting fluid amount per 12 mm cover glass created 7 mm diameter,circle shaped, thin liquid films containing the beads sandwiched betweenthe cover glass and the microarray substrate. The microarray substrateswere then placed on a UV transilluminator light box (TMW-20Transilluminator; Model White/UV; UVP, Upland, Calif.) and thesubstrates raised, by their edges, off the glass surface of the lightbox using ˜1 mm thick wetted filter papers (wetted to reduce evaporationof bead solutions). The light box was then covered. Prior to powering onthe light source, the substrates were allowed to stand, undisturbed, for5 min to allow equilibration. The substrates were then UV illuminated,from the bottom up, through the glass microarray substrate material for5 min without disturbance. After UV illumination, the substrates werethen left to stand for an additional 10 min without disturbance to allowbinding of the photo-released material to the epoxy activated substratesurface. To remove the cover glasses and wash away the beads, thesubstrates were dropped, face up, into an already-mixing tray of 5% BSA(w/v) in TBS-T and mixed for 1 min on an orbital platform shaker. Themicroarray substrates were additionally washed 4×30 sec with TBS-T and4×30 sec with purified water. To confirm that the beads were indeedwashed away, the microarray substrates were viewed under a standardphase contrast microscope (note: when present, the 10 micron diameterbeads are easily and clearly visible under the microscope). Thesubstrates were dried prior to imaging.

Detection of Photo-Transferred Protein:

Detection of the Cy5 fluorescence labeling in the photo-transferredcasein spots was achieved by imaging the dry microarray substrates on anArrayWoRx^(e) BioChip fluorescence reader (Applied Precision, LLC,Issaquah, Wash.) with the appropriate standard manufacturer suppliedfilter set. The optical scanning resolution was set to 3 microns.

Results:

One major advantage of the contact photo-transfer method is the abilityto print very high density microarrays, dictated by the bead size, todensities beyond what is possible with conventional mechanical printinginstruments. Additionally, unlike conventional mechanical printing,contact photo-transfer is not serial but fully parallel and thusprinting time and effort is independent of the number of array features,requiring only 5 min of illumination with the proper light. Mechanicalwear-and-tear of conventional robotic printing devices is alsoeliminated.

Photo-transferred spot diameters were measured using the softwaresupplied by Applied Precision, LLC (Issaquah, Wash.) with theirArrayWoRx^(e) BioChip reader. Spots in the entire 7 mm diameter printedarea were enumerated using simple 2-D electrophoresis spot detectionsoftware (ImageQuant; Molecular Dynamics; Amersham Biosciences Corp.,Piscataway, N.J.). As shown in FIG. 24, sharp, easily resolved, 13micron, circular microarray features were generated with this contractphoto-transfer method. “+PC-Casein” in FIG. 24 refers to the addition ofthe PC-biotin-casein-Cy5 conjugate to either the BSA coated negativecontrol beads (“BSA Bead” in FIG. 24) or the NeutrAvidin coated beads(“Avidin Beads” in FIG. 24) prior to the contact photo-transfer process.Spot signal is only observed when NeutrAvidin coated beads were used tocapture the PC-biotin-casein-Cy5 conjugate and only when UV lightirradiation was used to photo-release and transfer the conjugate to themicroarray substrate surface (“+hν” in FIG. 24). When negative controlBSA coated beads were used for capture of the PC-biotin-casein-Cy5conjugate, no signal was observed on the microarray since no conjugatewas specifically captured by its PC-biotin label. Observation under amicroscope confirmed that the beads were indeed washed away from themicroarray substrate prior to imaging. An additional negative control,performed from the NeutrAvidin beads loaded with thePC-biotin-casein-Cy5 conjugate, but without the UV light treatmentduring the contact photo-transfer process (“−hν” in FIG. 24), also showsno measurable signal. This negative control further confirms that thebeads are not bound to the array surface, but are indeed washed awaysince no signal from the bead-bound fluorescent casein conjugate wasobserved. At the spot density used, 4,896 spots were counted in the 7 mmcircular area which would correspond to 242,004 spots on an entire 25×75mm microarray substrate (129 spots/mm²).

Example 28 Contact Photo-Transfer for Molecular Diagnostic Assays:Cell-Free Expression from a PCR Template of the APC Gene Amplified fromGenomic DNA Preparation of a Photocleavable Antibody Affinity Matrix:

The photocleavable antibody beaded affinity matrix was prepared usingthe monoclonal anti-HSV tag antibody (EMD Biosciences, Inc., San Diego,Calif.) as described in Example 2.

PCR Amplification of an APC Segmentfrom Genomic DNA:

Isolation of genomic DNA from cultured cells and PCR amplification ofsegment 3 of the human APC gene was performed as reported by AmberGen inthe scientific literature [Gite et al. (2003) Nat Biotechnol 21,194-197], except that an N-terminal HSV epitope tag (amino acid sequenceQPELAPEDPED [SEQ NO. 3]) and an N-terminal VSV-G epitope tag wasincorporated into the expressed protein, instead of the N-terminal VSV-Gtag alone. The C-terminal p53 derived epitope tag was as previouslyreported [Gite et al. (2003) Nat Biotechnol 21, 194-197]. Epitope tagsand elements necessary for efficient cell-free expression are introducedinto the PCR amplicon by way of specialized primers [Gite et al. (2003)Nat Biotechnol 21, 194-197]. APC segment 3 of Exon 15 corresponds tocodons 1,099 to 1,696. Wild-type (WT) APC was derived from the HeLa cellline and the mutant, containing a chain truncation mutation at codon1,338 of APC (CAg→TAg), was derived from the SW480 cell line. The exactprimers used are listed below:

APC Segment 3 Forward Primer: [SEQ NO. 4]5′ggATCCTAATACgACTCACTATAgggAgACCACCATgTACACCgACATCgAgATgAACCgCCTgggCAAgggAggACAgCCTgAACTCgCTCCAgAggATCCggAAgATgTTTCTCCATACAggTCACggggA3′ APC Segment 3 Reverse Primer: [SEQNO. 5] 5′TTATTACAgCAgCTTgTgCAggTCgCTgAAggTACTTCTgCCTTCTgT AggAATggTATC3′Cell-Free Expression and tRNA Mediated Labeling:

The APC segment 3 was expressed in a cell-free reaction as describedearlier in Example 1 with the following exceptions: Only AmberGen'sBODIPY-FL-tRNA^(COMPLETE) was used at 4 μM for labeling and not thePC-biotin-tRNA^(COMPLETE) or any other misaminoacylated tRNA labelingreagents. The expression reaction size was only 20 μL for each sample.Instead of adding expressible purified plasmid DNA for translation, 1 μLof crude PCR amplified APC segment 3 DNA was added. Importantly, theaforementioned anti-HSV tag photocleavable antibody affinity beads wereadded directly into the expression reaction, at the start of theexpression reaction, as the last component. To do this, 10 μL of beadvolume was washed 2×400 μL briefly (briefly=5 sec vortex mix) with 0.1%BSA (w/v) in PBS. Washes were performed in a polypropylene 0.5 mLmicro-centrifuge tube and the beads pelleted in a micro-centrifuge. Thewashed bead pellet was then diluted to a 50% bead suspension (v/v) withthe same buffer. 2 μL of this 50% bead suspension was added to thecell-free expression reaction mixture as the last component. TranslationDilution Buffer (TDB) used to stop the reaction and prepare the samplecontained no protein carriers, BSA or otherwise, contained 10 mM DTTinstead of 2 mM and additionally contained 20 mM EDTA added from a 500mM pH 8.0 stock and 4 mM cycloheximide (Sigma-Aldrich, St. Louis, Mo.)added from a 355 mM stock in DMSO. The stopped translations alreadycontaining the beads were not equilibrated nor clarified as done inExample 1, but were instead processed for protein isolation as describedbelow.

Isolation of Labeled Nascent Proteins:

All steps were performed at +4° C. or on an ice water bath and allreagents and samples were also kept under these conditions during theprocedure. The bead suspension was gently mixed for 30 min to furtherallow capture of the nascent protein on the anti-HSV tag photocleavableantibody affinity beads. The bead suspensions were then diluted to 400μL final volume using 5 mM DTT in PBS. The beads were then washed 4× in400 μL of 5 mM DTT in PBS per wash. The first 2 washes were by 5 secvortex mixing and the last 2 washes for 5 min with gently mixing. Thebeads were then additionally washed 1× briefly (briefly=5 sec vortexmix) in 400 μL of 50% glycerol and 5 mM DTT in PBS. All washingprocedures were performed in batch mode using 0.45 micron pore size,PVDF membrane, micro-centrifuge Filtration Devices to facilitatemanipulation of the small volumes of affinity matrix (˜100 micron beads)and exchange the buffers (Utrafree-MC Durapore Micro-centrifugeFiltration Devices, 400 μL capacity; Millipore, Billerica, Mass.). Priorto contact photo-transfer of the captured and isolated proteins, thewashed pellet of 1 μL of beads was suspended in a final volume of 100 μLwith 50% glycerol and 5 mM DTT in PBS thereby resulting in a 1% (v/v)bead suspension that can be stored long-term at −20° C. without freezingof the sample and thus without damage to the agarose beads.

Contact Photo-Transfer from Individually Resolved Beads:

Contact photo-transfer from individually resolved beads onto epoxyactivated microarray substrates using 1 μL droplets of the 1% beadsuspension was performed as described in Example 14.

Detection of Photo-Transferred Protein:

Detection of the directly incorporated tRNA mediated BODIPY-FLfluorescence labeling in the photo-transferred APC segment 3 proteinswas achieved by imaging the dry microarray substrates on anArrayWoRx^(e) BioChip fluorescence reader (Applied Precision, LLC,Issaquah, Wash.) using the appropriate manufacturer supplied standardfilter set and the resolution set to 9.7 microns.

Results:

The results in FIG. 25 show that bead-derived 100 micron diameterprotein spots are clearly visible for the contact-photo transferredsegment 3 of the expressed human APC gene. Phase contrast microscopyconfirmed that the easily visible 100 micron beads are indeed washedaway from the microarray substrate following contact photo-transfer. Thesignal from the contact photo-transferred APC protein arises from theinternal tRNA mediated BODIPY-FL labels. A negative control, wherebyonly the needed PCR derived expression DNA was omitted from thecell-free translation step, shows no detectible signal on the microarraysubstrate. Fluorescently labeled antibodies directed against thegenetically engineered N- and C-terminal tags (introduced by PCRprimers) can also be used to detect the relative amount of truncated APCprotein for diagnostic purposes, analogous to detection withenzyme-labeled antibodies in an ELISA based colorectal cancer diagnosticassay [Gite et al. (2003) Nat Biotechnol 21, 194-197]. In an alternativemethod, the bead isolated APC protein can be contact photo-transferredto plain, activated (e.g. epoxy or aldehyde) or coated (e.g. hydrophobiccoatings or highly charged primary amine coatings) MALDI-TOF massspectrometry targets. In this case, the mutational or truncation statusof the protein is based on molecular weight as determined by massspectrometry analysis.

Example 29 Contact Photo-Transfer of Allergens to Microarrays for InVitro Diagnostics Conjugating Photocleavable Biotin & Cy5 to the CaseinTest Allergen:

Performed as described in Example 27.

Loading the PC-Biotin-Casein-Cy5 Conjugate to NeutrAvidin Coated AgaroseBeads:

The PC-biotin-casein-Cy5 conjugate is loaded onto 6% cross-linkedNeutrAvidin agarose beads (Pierce Biotechnology, Inc., Rockford, Ill.).This was done using 0.45 micron pore size, PVDF membrane,micro-centrifuge Filtration Devices to facilitate manipulation of thesmall volumes of affinity matrix (˜100 micron beads) and exchange thebuffers (Ultrafree-MC Durapore Micro-centrifuge Filtration Devices, 400μL capacity; Millipore, Billerica, Mass.). 50 μL of bead volume waswashed 4×400 μL with 1% BSA (w/v) in PBS. The PC-biotin-casein-Cy5conjugate was then diluted to 12.5 μg/mL with 1% BSA (w/v) in PBS. Thediluted conjugate solution was then added to the washed bead pellet andthe resultant suspension was gently mixed for 30 min to allow binding.Based on measuring the visible absorbance spectrum of the Cy5 componentof the conjugate (λ_(max)=649 nm; molar extinction coefficient=250,000)in a spectrophotometer, 94% of the added conjugate was captured on thebeads. The beads were then washed 2×400 μL with 1% BSA (w/v) in PBSfollowed by 2×400 μL for 1 min each with 10 mM d-biotin dissolved in a200 mM sodium bicarbonate and 200 nM NaCl buffer, in order to quench theremaining biotin binding sites on the beads. Lastly the beads werewashed 1×400 μL with 50% glycerol (v/v) and 5 mM DTT in PBS. All washeswere for 5 sec vortex mixing followed by filtration. The beads wereresuspended to a 10% (v/v) suspension with 50% glycerol (v/v) and 5 mMDTT in PBS.

Contact Photo-Transfer Based Microarray Assays for Detection ofAllergen-Specific IgE in Human Sera from Allergy Patients:

2 distinct assay formats are demonstrated in this Example: For format#1, the aforementioned prepared bead-bound PC-biotin-casein-Cy5 allergenconjugate is first contact photo-transferred to the microarray substrateand the entire allergen-specific IgE assay performed on the microarrayitself. For format #2, the allergen-specific IgE assay is performed onthe beads prior to contact photo-transferring the bound material to amicroarray substrate for readout.

As mentioned above, for format #1, the bead-bound PC-biotin-casein-Cy5allergen conjugate must first be contact photo-transferred to themicroarray substrate. The contact photo-transfer process was performedas described in Example 24, except that beads were contactphoto-transferred over nearly an entire 25×75 mm microarray substrate,instead of a 12 mm diameter circular region. For this, 2 μL bead volumeof the aforementioned prepared PC-biotin-casein-Cy5 beads was washed1×400 μL with 50% glycerol (v/v) and 5 mM DTT in PBS and resuspended toa 1% (v/v) suspension with the same buffer. 100 μL of the beadsuspension was applied to the microarray substrate and overlaid with a22×60 mm rectangular microscope cover glass for the contactphoto-transfer process. Following contact photo-transfer, the microarraysubstrate was washed 4×30 sec with excess TBS-T (cover glass removed)followed by 4×30 sec with purified water. Phase contrast microscopyverifies that the easily visible ˜100 micron diameter beads were indeedwashed away from the microarray surface. The microarray substrate wasdried prior to usage in the allergen assay.

For performing the allergen assay on the microarray (format #1), theaforementioned spotted microarray substrate was subdivided into 16 wellsusing a commercially available gasket overlay system (ProPlate™Multiarray Slide System; Grace Bio-Labs, Inc., Bend, Oreg.). The wellswere pre-blocked for 1 hr with excess 5% BSA (w/v) in PBS-T [PBS with0.05% Tween-20 (v/v)]. The wells were then treated with 50 μL of acommercially available verified human serum from a patient with a caseindependant milk allergy (PlasmaLab, Everett, Wash.). 1× concentratedserum or 1/10 diluted serum was used. As a negative control, a separatewell was treated with serum from a non-allergic patient. The treatmentwas performed for 2 hr with gentle mixing to allow binding of theallergen-specific IgE. The wells were then washed 3× with excess PBS-T.The microarray-bound IgE was then detected with 50 μL/well ofanti-[human IgE] polyclonal antibody (Bethyl Laboratories, Montgomery,Tex.) conjugated to the Cy3 fluorophore (Amersham Biosciences Corp.,Piscataway, N.J.) and diluted to 0.5 μg/mL with 5% BSA (w/v) in PBS-T.Detection was performed for 1 hr with gentle mixing. The wells were thenwashed 3× with excess PBS-T and then 3× with excess purified water. Themicroarray substrates were dried prior to imaging.

For performing the allergen assay on the beads (format #2), the beadswere first pre-blocked by washing 3× with excess 5% BSA (w/v) in PBS-Tusing the aforementioned micro-centrifuge Filtration Devices. The beadswere then treated with 100 μL of a commercially available verified humanserum from a patient with a casein dependant milk allergy (PlasmaLab,Everett, Wash.). As a negative control, a second set of beads wastreated with serum from a non-allergic patient. The treatment wasperformed for 2 hr with gentle mixing to allow binding of theallergen-specific IgE. The beads were then washed 3× with excess PBS-Tusing the aforementioned micro-centrifuge Filtration Devices. Thebead-bound IgE was then detected with an anti-[human IgE] polyclonalantibody (Bethyl Laboratories, Montgomery, Tex.) conjugated to the Cy3fluorophore (Amersham Biosciences Corp., Piscataway, N.J.) and dilutedto 0.5 μg/mL with 5% BSA (w/v) in PBS-T. Detection was performed for 1hr with gentle mixing. The beads were then washed 3× with excess PBSusing the aforementioned micro-centrifuge Filtration Devices and thendiluted with 50% glycerol (v/v) and 5 mM DTT in PBS to a 1% (v/v) beadsuspension. Contact photo-transfer was then performed as described inExample 24. Following contact photo-transfer, the microarray substratewas washed 4×30 sec with excess PBS-T (cover glasses removed) followedby 4×30 sec with purified water. Phase contrast microscopy verifies thatthe easily visible ˜100 micron diameter beads were indeed washed awayfrom the microarray surface. The microarray substrates were dried priorto imaging.

Detection of Photo-Transferred Protein.

Detection of the Cy5 fluorescence labeling in the photo-transferredcasein spots as well as the Cy3 fluorescence for measuring the boundallergen-specific IgE was achieved by imaging the dry microarraysubstrates on an ArrayWoRXe BioChip fluorescence reader (AppliedPrecision, LLC, Issaquah, Wash.) with the appropriate standardmanufacturer supplied filter sets.

Results:

Results are shown in FIGS. 26A and B. FIG. 26A (format #1), where theallergen-specific IgE assay was performed on casein that was firstphoto-transferred to the microarray, shows specific detection of thecasein-directed IgE in the 1× and 1/10× diluted test serum (“MilkAllergy Serum”) as compared to the blank corresponding to 1× serum froma non-allergic patient (“Negative Serum”). FIG. 26B (format #2), wherethe allergen-specific IgE assay was performed on the casein-containingbeads followed by contact photo-transfer, also shows specific detectionof the casein-directed IgE in the 1× test serum as compared to theblank. The slight background signal in the blank samples was determinedto arise from fluorescence cross-talk of the intense Cy5 signal in thedirectly labeled casein conjugate into the Cy3 fluorescence channel ofthe microarray reader, and not non-specific detection of IgE. Thisproblem can be solved by better fluorescence filtering or lowering theCy5 labeling ratio of the casein conjugate.

Example 30 Solid-Phase Bridge PCR and Cell-Free Expression of theSolid-Phase Bridge PCR Amplicon Preparation of Beads CovalentlyConjugated to PCR Primers:

The following forward and reverse PCR primers were purchased fromSigma-Genosys (The Woodlands, Tex.), both with a 5′ primary aminemodification following a 6 carbon spacer (see later in this Example forcorresponding template):

[SEQ NO. 6] Forward: 5′[Amine]CgTCCCgCgAAATTAATACgACTCAC3′ [SEQ NO. 7]Reverse: 5′[Amine]gTTAAATTgCTAACgCAgTCAggAg3′Primary amine reactive, NHS ester activated (N-hydroxysuccinimide), 4%cross-linked agarose beads (˜100 micron diameter) were purchased fromAmersham Biosciences (Amersham Biosciences Corp., Piscataway, N.J.). 100μL bead volume was spun down in a micro-centrifuge and the isopropanolstorage buffer removed. The remaining procedures, unless otherwisenoted, were performed in batch mode using 0.45 micron pore size, PVDFmembrane, micro-centrifuge Filtration Devices to facilitate manipulationof the beaded matrix (˜100 micron beads) and exchange the buffers(Ultrafree-MC Durapore Micro-centrifuge Filtration Devices, 400 μLcapacity; Millipore, Billerica, Mass.). The 100 μL of beads were washed3× briefly (briefly=5 sec vortex mix) with 400 μL each of ice cold 1 mMHCl prepared in nuclease-free water. The washed bead pellet was thenresuspend in 200 μL containing 100 μg of each primer (forward andreverse) prepared in 200 mM sodium bicarbonate and 2M NaCl (allnuclease-free). As a negative control, a second set of beads receivedplain buffer only (without primers). The binding reaction was allowed toproceed for 1 hour at room temperature with gentle mixing. The beadswere then washed 1× briefly (briefly=5 sec vortex mix) with 400 μL of200 mM sodium bicarbonate, 200 mM glycine, 1 mM EDTA and 2M NaCl (allnuclease-free) and then the remaining reactive sites were quenched byadding 2×400 μL of the same buffer for 30 min each with gentle mixing.The beads were then washed 2× briefly (briefly=5 sec vortex mix) with200 mM sodium bicarbonate and 2M NaCl (all nuclease-free) followed by2×5 min each with 10 mM Tris, pH 8.0, 2M NaCl and 1 mM EDTA (allnuclease-free). Beads were lastly washed 1× briefly (briefly=5 secvortex mix) with 50% glycerol, 5 mM Tris, pH 8.0, 2M NaCl and 0.5 mMEDTA (all nuclease-free) and then diluted to a 20% (v/v) bead suspensionin the same buffer. This bead stock was stored in a 0.5 mL PCR tube at−20° C.

Qualitative Analysis of Primer Attachment:

To verify successful primer attachment to the beads, an aliquot of thebeads was stained with the single-stranded DNA fluorescence-baseddetection reagent OliGreen (Invitrogen Corporation, Carlsbad, Calif.).This reagent is essentially non-fluorescent until bound tosingle-stranded DNA at which point it can be imaged using any standardfluorescein-type fluorescence detection system. The manufacturersupplied reagent was diluted 1/200 in 10 mM Tris, pH 8.0, 1 mM EDTA and0.01% (v/v) Tween-20 (all nuclease-free). 20 μL of the preparedprimer-conjugated bead stock (20% beads for 4 μL actual bead volume) wasmixed with 100 μL of the diluted OliGreen reagent in a thin-walled 0.5mL polypropylene clear PCR tube. As a negative control, beads that wereprepared the same except lacked any attached primer were also tested.After approximately 1 min, the beads were spun down in amicro-centrifuge and imaged directly in the tubes using a FluorImager SIlaser-based fluorescence scanner (488 nm argon laser excitation and 530nm emissions filter) (Molecular Dynamics/Amersham Biosciences Corp.,Piscataway, N.J.).

Preparation of the Template for Solid-Phase Bridge PCR:

The template DNA for the solid-phase bridge PCR reaction was a linearDNA construct corresponding to the human glutathione-s-transferase A2gene (GST A2; open reading frame additionally containing epitope tagsequences and untranslated sequences needed for efficient cell-freeexpression). Using an initial solution-phase PCR reaction (same primersas above in this Example, listed again below), this linear DNA constructitself was created from GST A2 that was cloned into the cell-freeexpressible pETBlue-2 plasmid (EMD Biosciences, Inc., San Diego, Calif.)(see Example 1 for cloning). The solution-phase PCR was performedaccording to standard practices and using a commercially available kitaccording to the manufacturer's instructions (SuperTaq™ DNA PolymeraseKit; Ambion, Austin, Tex.). Prior to serving as the template forsubsequent solid-phase bridge PCR, the solution-phase PCR amplicon(product) (i.e. the linear DNA construct) was first purified andconcentrated on silica-based columns using a commercially available kitaccording to the manufacturer's instructions (QIAquick PCR PurificationKit; Qiagen, Valencia, Calif.).

Solution-Phase Primers: Forward: 5′CgTCCCgCgAAATTAATACgACTCAC3′ [SEQ NO.8] Reverse: 5′gTTAAATTgCTAACgCAgTCAggAg3′ [SEQ NO. 9]Solid-Phase Bridge PCR with Primer-Conjugated Beads:

Solid-phase “Bridge” PCR was originally developed and patented by Adamsand Kron [U.S. Pat. No. 5,641,658] and is used for multiplexed geneticanalyses on various solid-surfaces including beads. The mechanism ofsolid-phase bridge PCR is reported by Adams and Kron [U.S. Pat. No.5,641,658] as well as in the scientific literature [Tillib et al. (2001)Anal Biochem 292, 155-160; Shapero et al. (2001) Genome Res 11,1926-1934; Mitterer et al. (2004) J Clin Microbiol 42, 1048-1057; Adessiet al. (2000) Nucleic Acids Res 28, E87]. In this Example, immobilizedDNA produced by solid-phase bridge PCR is ultimately used as a templatefor cell-free protein expression.

In this Example, for solid-phase bridge PCR, 5 μL bead volume of theprimer-conjugated agarose beads was washed 4×400 μL with nuclease-freewater using the 0.45 micron pore size, PVDF membrane, micro-centrifugeFiltration Devices (Ultrafree-MC Durapore Micro-centrifuge FiltrationDevices, 400 μL capacity; Millipore, Billerica, Mass.). As a negativecontrol, 5 μL of beads which lack any bound primer (see earlier in thisExample for beads) was washed in the same manner. 50 μL of prepared PCRreaction mixture was used to resuspend the washed bead pellets whichwere then transferred to PCR tubes for thermocycling. Solid-Phase bridgePCR was performed essentially using standard solution-phase PCRpractices and a commercially available kit according to themanufacturer's instructions (SuperTaq™ DNA Polymerase Kit; Ambion,Austin, Tex.). However, no soluble primers were added at any step. Thehuman GST A2 template DNA (see earlier in this Example) was used at 10ng per 50 μL of PCR reaction. The following thermocycling steps wereused for the solid-phase bridge PCR reaction: Initially 94° C. 2 min(once) and then 60 cycles of 94° C. 30 s, 60° C. 30 s and 72° C. 2 min,followed by a final 72° C. 10 min (once).

Expression from the Solid-Phase Bridge PCR Beads:

Following the solid-phase bridge PCR reactions, the 5 μL of beads fromeach PCR reaction sample was then washed 3× with 400 μL each of nucleasefree water using the 0.45 micron pore size, PVDF membrane,micro-centrifuge Filtration Devices (Ultrafree-MC DuraporeMicro-centrifuge Filtration Devices, 400 μL capacity; Millipore,Billerica, Mass.). Each washed bead pellet was resuspended in 40 μL ofcomplete rabbit reticulocyte cell-free expression mixture and expressedas described in Example 1 with the following exceptions: For expressionof solid-phase bridge PCR beads, no soluble DNA was included in thereaction. The reaction mixture containing the beads was gently shakenthroughout the expression procedure. Control samples were also performedby expressing soluble plasmid DNA, without any solid-phase bridge PCRbeads, as described in Example 1. In all cases, for fluorescent labelingof the nascent protein, only BODIPY-FL-tRNA^(COMPLETE) was included inthe reaction at 2 μM final and no other added misaminoacylated tRNAswere used. The crude expression reactions, with and without beads, wereprocessed for and analyzed using standard denaturing SDS-PAGE followedby imaging of the fluorescent BODIPY labels using a FluorImager SIlaser-based gel scanner (Molecular Dynamics/Amersham Biosciences Corp.,Piscataway, N.J.).

Results:

The results are shown in FIGS. 27A and 27B. FIG. 27A shows the resultsof qualitative verification of PCR primer attachment to the activatedagarose beads. The fluorescent OliGreen DNA detection reagent showssignificant positive signal on the beads (“Bead Pellet”) in the casewhere the beads were loaded with amine functionalized PCR primers(“Primer Beads”) and negligible background signal from the beads wherethe PCR primers were omitted in the conjugation reaction (“BlankBeads”). Quantification of the signal shows a 100:1 signal to background(blank) ratio.

FIG. 27B shows fluorescence SDS-PAGE analysis of the cell-freeexpression of human glutathione-s-transferase A2 from the bead-boundfull length DNA which was created by the solid-phase bridge PCR reaction(Lane 3 in the Figure; arrows indicate expressed and labeled nascentprotein). As a negative control, beads lacking any bound primers forsolid-phase bridge PCR produced no glutathione-s-transferase A2 in thecell-free expression reaction (Lane 4 in Figure). For comparison,positive controls corresponding to human p53 protein (Lane 1 in Figure)and human glutathione-s-transferase A2 (Lane 2 in Figure) that werecell-free expressed from soluble plasmid DNA, without any beads, usingstandard procedures as described in Example 1. Quantification of thefluorescent protein bands on the SDS-PAGE gel show that the bead-boundglutathione-s-transferase A2 DNA created by solid-phase bridge PCRexpresses 2-fold less than the standard soluble plasmidglutathione-s-transferase A2 DNA.

Example 31 Multiplex Solid-Phase Bridge PCR Followed by MultiplexCell-Free Expression with In Situ Protein Capture and ContactPhoto-Transfer to Microarray Surfaces Preparation of Beads CovalentlyConjugated to PCR Primers.

Conjugation of 5′ amine modified PCR primers to agarose beads wasperformed as described in Example 30 with the following exceptions: 2batches of beads were prepared containing 2 different sets ofgene-specific PCR primers. The PCR primers also contained elementsnecessary for efficient cell-free expression (T7 promoter and Kozaksequence) as well as a C-terminal HSV epitope tag. Primers sets forhuman p53 and γ-actin genes were used and were as follows:

γ-Actin Forward: [SEQ NO. 10]5′[Amine]ggATCCTAATACgACTCACTATAgggAgCCACCATggAAgAAgAgATCgCCgCgCTggTCATTgAC3′ γ-Actin Reverse: [SEQ NO. 11]5′[Amine]TTAATCCTCTgggTCTTCAggAgCgAgTTCTggCTggCTgAAgCATTTgCggTggACgATggAggggCC3′ p53 Forward: [SEQ NO. 12]5′[Amine]ggATCCTAATACgACTCACTATAgggAgACCACCATggAgg AgCCgCAgTCAgATCCT3′p53 Reverse: [SEQ NO. 13]5′[Amine]TTTTAATCCTCTgggTCTTCAggAgCgAgTTCTggCTggCTgTCTgAgTCAggCCCTTCTgTC3′

Additionally, during the conjugation of primers to the agarose beads, abiotin-amine linker (EZ-Link Amine-PEO3-Biotin; Pierce Biotechnology,Inc., Rockford, Ill.) was incorporated into the reaction mixture alongwith the primers. This was achieved by diluting a 20 mg/mL biotin-aminelinker stock (prepared in nuclease-free water) 1/100 in nuclease-freewater and adding 5 μL to the primer reaction mixture described inExample 30 (biotin-amine linker added prior to adding primer reactionmixture to beads). This level of biotin-amine linker constituted 10-foldless moles relative to the total primer amount. 100-fold less moles ofbiotin-amine linker can also be used with success.

Qualitative Analysis of Primer Attachment:

Performed as in Example 30.

Solid-Phase Bridge PCR with Primer-Conjugated Beads:

Performed essentially as in Example 30 with the following exceptions:For solid-phase bridge PCR, 2.5 μL bead volume of each bead-bound primerset (p53 and γ-actin; 5 μL bead volume total) was washed 3×400 μL withnuclease-free water, the two different bead species (p53 and γ-actin)were combined and the pellet resuspended in 50 μL of prepared PCRreaction mixture. A single multiplexed solid-phase bridge PCR reactionwas performed on the pooled bead species using standard PCR reagents(SuperTaq™ DNA Polymerase Kit; Ambion, Austin, Tex.) and a HeLa cellcDNA library as template. The cDNA template was prepared by extractingtotal RNA from cultured HeLa cells using a commercially available kitaccording to the manufacturer's instructions (RNeasy Maxi; Qiagen,Valencia, Calif.). mRNA was then isolated from the total RNA using acommercial kit according to the manufacturer's instructions (Oligotex;Qiagen, Valencia, Calif.). mRNA was then converted to cDNA usingstandard RT-PCR practices (e.g. using Omniscript RT-PCR kit; Qiagen,Valencia, Calif.). Alternatively, the total RNA can be converteddirectly to cDNA via RT-PCR instead of using purified mRNA. Forsolid-phase bridge PCR with the HeLa cell cDNA template, thermocyclingwas as follows: Initially 94° C. 2 min and then 60 cycles of 94° C. 30s, 60° C. 30 s and 72° C. 2 min, followed by a final 72° C. 10 min.

Attaching the PC-Antibody to Beads Following Solid-Phase Bridge PCR:

Following the solid-phase bridge PCR reaction, beads were washed briefly3× with nuclease-free water and 1× with TE-Saline (10 mM Tris-HCl, pH8.0, 1 mM EDTA, 200 mM NaCl), all at 400 μL (all nuclease-freereagents). Unless otherwise noted, all washes and bead manipulationswere performed in batch mode using 0.45 micron pore size, PVDF membrane,micro-centrifuge Filtration Devices to facilitate manipulation of thebeaded matrix (˜100 micron beads) and exchange the buffers (Ultrafree-MCDurapore Micro-centrifuge Filtration Devices, 400 μL capacity;Millipore, Billerica, Mass.). NeutrAvidin (tetrameric) was then attachedto the bead bound biotin-amine linker (see earlier in this Example), inexcess, by treatment with 200 μL of a 0.5 μg/μL solution in TE-Salinefor 30 min. Beads were washed briefly 4×400 μL with TE-Saline.

The beads were next coated with a polyclonal rabbit anti-HSV tag captureantibody (Bethyl Laboratories, Montgomery, Tex.) which was converted tophotocleavable form by conjugation to PC-biotin. Creation of thephotocleavable antibody (PC-antibody) was performed similar to asdescribed in Example 2. To first create the PC-antibody (prepared inadvance), 400 μg of antibody as supplied by the manufacturer at 1 μg/μLwas purified on a NAP-5 desalting column according to the manufacturer'sinstructions (Amersham Biosciences Corp., Piscataway, N.J.) against a200 mM sodium bicarbonate and 200 mM NaCl buffer (nuclease-freereagents). The resultant antibody was then reacted with 20 molarequivalents of AmberGen's PC-biotin-NHS labeling reagent (added from a50 mM stock in anhydrous DMF) (15 to 25 molar equivalents can also beused) for 30-60 min with gentle mixing. The labeled antibody was thenpurified on a NAP-10 desalting column according to the manufacturer'sinstructions (Amersham Biosciences Corp., Piscataway, N.J.) againstTE-Saline buffer. This prepared polyclonal anti-HSV PC-biotin conjugatewas then loaded onto the beads by treatment of the beads with 150 μL of0.15 μg/μL in TE-Saline for 30 min. Beads were washed briefly 4× and1×30 min (30° C.) in 400 μL TE-Saline followed by 2× brief washes innuclease-free water.

Multiplexed Cell-Free Expression of the Beads and In Situ ProteinCapture:

The 5 μL bead pellet was then resuspended in 50-100 μL of completerabbit reticulocyte cell-free expression mixture and expressedessentially as described in Example 1 with the following exceptions: Nosoluble DNA was included in the reaction and tRNA mediated labeling waswith 2 μM BODIPY-FL-tRNA^(COMPLETE) only (i.e. no tRNA mediatedPC-biotin labeling). To disperse the beads and limit diffusion during insitu capture, the expression mixture was spread over the surface of aplain glass microscope slide and overlaid with a cover glass (seeExamples 25 and 26 for mechanism and details of in situ capture). Asdetailed earlier, in situ capture was mediated by a common C-terminalHSV epitope tag in all expressed proteins and the anti-HSV PC-antibodyon the beads. Expression was carried out in a humidified chamber. Afterexpression, the microscope slide (and cover glass) “sandwich” was placedin a 50 mL polypropylene centrifuge tube and sprayed at the seam with300 μL of TDB supplemented with 1% BSA (w/v) as the protein carrier and10 μg of the soluble unlabeled monoclonal anti-HSV antibody. The beadsand fluid were then recovered by brief spinning in a clinicalcentrifuge. Beads were then immediately washed 2× briefly and 2×5 mineach with 400 μL of ice cold 5 mM DTT in PBS per wash. The beads werethen washed 1× briefly in 400 μL of 40% glycerol, 5 mM DTT in PBS. Allwashes were performed using the aforementioned micro-centrifugeFiltration Devices. The washed bead pellets were then resuspended to 1%beads (v/v) in 40% glycerol, 5 mM DTT in PBS.

Contact Photo-Transfer from Individually Resolved Beads:

Contact photo-transfer from individually resolved beads onto epoxyactivated glass microarray substrates (slides) (SuperEpoxy substrates,TeleChem International, Inc. ArrayIt™ Division, Sunnyvale, Calif.)overlaid with a cover glass was performed as described in Example 24;except that after contact photo-transfer, washing and drying of themicroarray slides, the slides were further processed for antibodyprobing as described in the following paragraphs.

Preparation of an Anti-p53 Cy5 Fluorescent Antibody:

Performed as described in Example 26.

Probing the Microarray with Anti-p53-Cy5 Antibody:

Performed as described in Example 26. Alternatively, a modification tothe procedure can be used where the antibody probe is used at a 1/100dilution. In this case, probing is achieved by applying 100 μL ofdiluted antibody probe to the microarray slide and overlaying with a22×60 mm microscope cover glass (binding is then performed in ahumidified chamber).

Detection of Photo-Transferred Protein:

Performed as described in Example 26.

Results:

Fluorescence images of the same region of the same microarray slide areshown in FIG. 28. The green fluorescence channel shows the direct tRNAmediated BODIPY-FL labeling, allowing detection of the roughly 100micron diameter protein spots formed by contact photo-transfer,regardless of whether they are p53 or γ-actin. The red fluorescencechannel shows selective detection of the protein spots with theanti-p53-Cy5 antibody, in order to distinguish the p53 spots (red andgreen fluorescence signal) from the γ-actin spots (only greenfluorescence signal). Spots identified as p53 are marked by arrows inFIG. 28 in both the green and red fluorescence channels. Spotsidentified as γ-actin (unmarked; green signal only) show virtually nodetectible red fluorescence signal (p53 antibody), thus demonstrating nocross-contamination. These data clearly demonstrate that the p53 andγ-actin solid-phase bridge PCR amplicons (DNA) are indeed sorted (pure)on their respective (parent) primer-coated beads and hence, by way of insitu protein capture, the expressed proteins are also sorted on theirparent beads. The entire process is shown to be compatible with contactphoto-transfer fabrication of protein microarrays.

Example 32 Solid-Phase Bridge PCR on 7 Micron Diameter Non-PorousPlastic Beads: On-Bead DNA Detection Primer Attachment to 7 MicronDiameter Non-Porous Plastic Beads

mL of nuclease-free BSA (100 mg/mL; Invitrogen Corporation, Carlsbad,Calif.) was desalted on a NAP-5 column according to the manufacturer'sinstructions (Amersham Biosciences Corp., Piscataway, N.J.) versusConjugation Buffer (200 mM sodium bicarbonate and 200 mM NaCl). Therecovered BSA solution was 1 mL at 50 mg/mL. 8 mg of EZ-LinkSulfo-NHS-LC-LC-Biotin powder (Pierce Biotechnology, Inc., Rockford,Ill.) was then dissolved in 239 μL of nuclease-free water andimmediately after dissolving, 73 μL was added to the 1 mL of 50 mg/mLBSA. The reaction was carried out for 30 min at room temperature withgentle mixing. The biotinylated BSA was then desalted on NAP-5 columnsaccording to the manufacturer's instructions (Amersham BiosciencesCorp., Piscataway, N.J.) versus MES Buffer (0.1 M MES, pH 4.7, 0.9%NaCl; Pierce Biotechnology, Inc., Rockford, Ill.). The biotinylated BSAwas then diluted to 3.5 mg/mL in MES Buffer.

The biotinylated BSA solution was then used to coat commerciallyavailable 7.16 micron diameter non-porous amine-derivatized polymer(plastic) beads [catalog number PA06N;polymer=poly(MMA\GlycidylMethAcrylate\EDMA)+EDA; Bangs Laboratories,Inc. Fishers, Ind.]. To wash and manipulate the beads or exchange thebuffers, 0.45 micron pore size, PVDF membrane, micro-centrifugeFiltration Devices were used unless otherwise noted (Ultrafree-MCDurapore Micro-centrifuge Filtration Devices, 400 μL capacity;Millipore, Billerica, Mass.). A total 228 mg of beads (divided into 4equal aliquots) was washed 4×400 μL (each aliquot) with MES Buffer(unless otherwise noted, all washes are brief, 1-3 sec, by vortexmixing). The washed beads were then pooled in a single 1.5 mLmicro-centrifuge tube (all supernatant then removed) and the bead pellet(228 mg beads and about 200 μL bead volume) was then pre-chilled on anice water bath. 10 mg of EDC(1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride powder;Pierce Biotechnology, Inc., Rockford, Ill.) was then dissolved in 0.5 mLof ice-cold nuclease-free water. Immediately after dissolving, 100 μL ofthe EDC solution was added to the chilled bead pellet. 1 mL of roomtemperature biotinylated BSA solution (3.5 mg/mL in MES Buffer) was thenimmediately added to the bead-EDC mixture. The reaction was carried outfor 1 hr at room temperature with gentle mixing. Note that subsequentwashing of the beads in the 1.5 mL tube involved 1-3 sec vortex mixing,followed by pelleting the beads by micro-centrifugation at maximum speed(13,000 rpm) and discarding the supernatant. All buffers werenuclease-free. After the reaction, the beads were washed in the same 1.5mL tube at 2×1 mL using Quenching Buffer (200 mM glycine, 1 mM EDTA, 200mM sodium bicarbonate and 2M NaCl). The beads were then treated for 30min at room temperature with a fresh 1 mL of Quenching Buffer. Again inthe same tube, the beads were washed 2×1 mL with TE-NaCl-Glycine Buffer(10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 2M NaCl, 0.1M glycine). Afterre-suspension in the second wash, the beads were then pooled in one ofthe aforementioned Filtration Devices (using filtration to concentrateand pool beads). In the Filtration Device, beads were further washed4×400 μL with TE-Saline-Tween Buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA,200 mM NaCl, 0.1% Tween-20) and then 2×400 μL with TE-Glycerol-TweenBuffer (50% glycerol, 5 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 0.1%Tween-20). These biotin-BSA coated beads were then resuspend to 20%(v/v) beads (roughly 200 mg/mL beads) with TE-Glycerol-Tween Buffer andcould be stored at −20° C.

These biotin-BSA coated plastic beads were then conjugated to the 5′amine modified solid-phase bridge PCR primers. The primers were the sameas described in Example 30. The Working Primer Mix was prepared asfollows: A mixture of 100 μg of each amine modified primer (forward andreverse; 200 μg total primer) was freshly prepared in MES Buffer. To doso, 10 μL each of 10 μg/μL primer stocks (forward and reverse primerstocks; stocks prepared in nuclease-free water and stored at −20° C.)was mixed with 180 μL of MES Buffer, thus yielding 1 μg/μL total primerconcentration with 200 μg total primer in 90% MES Buffer (200 μL finalvolume).

Again using the aforementioned Filtration Devices, 25 μL of biotin-BSAcoated plastic bead volume was washed 4×400 μL with MES Buffer (unlessotherwise noted, all washes are brief, 1-3 sec, by vortex mixing).Directly in the Filtration Devices, to each bead pellet, 200 μL of thepreviously prepared Working Primer Mix was added. 10 mg of EDC was thenimmediately dissolved in 200 μL of ice cold nuclease-free water (50mg/mL EDC stock). Immediately after dissolving the EDC, 86 μL of EDCsolution was added to the primer-bead mix in the Filtration Device. Thereaction was carried out for 1 hr at room temperature in the upperchamber of the Filtration Device (without yet performing filtration)with mixing. In the Filtration Device, the beads were washed 1×400 μLwith TE-NaCl-Glycine Buffer and quenched by treatment for 30 min withmixing in a fresh 400 μL of the same buffer. Beads were then washed2×400 μL with TE-NaCl (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 2M NaCl), then2×400 μL TE-Saline-Tween and lastly 1×400 μL with TE-Glycerol-TweenBuffer. Beads were then resuspended to 20% (v/v) beads inTE-Glycerol-Tween Buffer and could be stored at −20° C.

Solid-Phase Bridge PCR on 7 Micron Diameter Non-Porous Plastic Beads

The prepared 7 micron diameter, biotin-BSA-primer coated beads were thenused for solid-phase bridge PCR as described in Example 30 with thefollowing exceptions: 2.5 μL bead volume was used in a 50 μL PCRreaction. Fluorescence BODIPY-FL-dUTP labeling of the solid-phase bridgePCR amplicon was performed by including the reagent (ChromaTide® BODIPY®FL-14-dUTP; Invitrogen Corporation, Carlsbad, Calif.) in the solid-phasebridge PCR reaction (20 μM final concentration added from themanufacturer's stock of 1 mM).

Fluorescence Imaging of Individual 7 Micron Diameter Non-Porous PlasticBeads:

The BODIPY-FL-dUTP labeling of the solid-phase bridge PCR amplicon onthe beads was imaged, at the individual bead level, by embedding thebeads in a thin polyacrylamide film on top of a microscope slide. Priorto embedding the beads, they were washed following the solid-phasebridge PCR reaction 3×400 μL with TE-Saline-Tween (10 mM Tris-HCl, pH8.0, 1 mM EDTA, 0.1% v/v Tween-20 and 200 mM NaCl; nuclease-free). Theacrylamide mix was prepared by mixing 487 μL TE (10 mM Tris-HCl, pH 8.0,1 mM EDTA; nuclease-free), 113 μL of a 40% acrylamide and bis-acrylamidemixture (19:1 ratio; Bio-Rad Laboratories, Hercules, Calif.), 1 μL of100% TEMED (Bio-Rad Laboratories, Hercules, Calif.) and 6 μL of a 10%(w/v) ammonium persulfate solution (prepared in water). This acrylamidemix was used to resuspend the washed bead pellet to form 1% (v/v) beads.Approximately 10-20 μL of the bead suspension was placed on a standardglass microscope slide, overlaid with an 18 mm round microscope coverglass and allowed to polymerize for approximately 10 min. The microscopeslides were fluorescently imaged as for other microarrays, such asdescribed in Example 26.

Results:

Results shown in FIG. 29 clearly show detection of the BODIPY-FL-dUTPlabeled solid-phase bridge PCR amplicon only when the necessary DNApolymerase was included in the solid-phase bridge PCR reaction (Plus DNAPolymerase). A separate solid-phase bridge PCR reaction, lacking onlythe necessary DNA polymerase (Minus DNA Polymerase), was performed toprovide the background levels related to bead auto-fluorescence and theBODIPY-FL-dUTP labeling reagent. A minus template negative controlsolid-phase bridge PCR reaction could also be used to assess backgroundlevels, with similar results. Upon quantification of the fluorescencesignal from several beads, the signal-to-noise ratio was determined tobe approximately 10:1.

Example 33 Solid-Phase Bridge PCR on 7 Micron Diameter Non-PorousPlastic Beads: Cell-Free Protein Expression, In Situ Protein Capture andBead Selection with Magnetic Particles Preparing Anti-[Mouse IgG]Species Specific Secondary Antibody Coated 1 Micron Diameter MagneticParticles

Secondary antibody coated 1 micron diameter magnetic particles werefirst prepared, in order to be used for isolation of 7 micron diameterplastic beads carrying primary antibody targeted cell-free expressedproteins. For this, amine-reactive p-toluensulphonyl chloride activated,1 micron diameter, magnetic particles (beads) were purchasedcommercially and coated with secondary antibody essentially according tothe magnetic particle manufacturer's instructions (Dynabeads® MyOne™Tosylactivated; Dynal Biotech LLC, Brown Deer, Wis.). The antibody was acommercially available donkey anti-[mouse IgG] species-specificsecondary antibody (Chemicon International, Inc., Temecula, Calif.;catalog number AP192). First, the antibody, as supplied by themanufacturer (0.5 mL at 2 mg/mL), was desalted on a NAP-5 column versusborate buffer (0.1M sodium tetraborate decahydrate, pH 9.5) according tothe column manufacturer's instructions (Amersham Biosciences Corp.,Piscataway, N.J.). The resultant antibody solution (0.54 μg/μL) was usedfor coating the magnetic particles. 12 mg of magnetic particles waspre-washed 2×1 mL with borate buffer. All washes were performed ineither 1.5 mL or 0.5 mL polypropylene micro-centrifuge tubes using acommercially available magnet (MPC-S magnet system; Dynal Biotech LLC,Brown Deer, Wis.) to draw the particles to the side-wall of the tubefollowed by removal of the fluid supernatant. The magnetic particleswere then resuspended to 100 μL total volume with borate buffer andmixed with 850 μL of the aforementioned prepared antibody solution (˜460μg antibody). 475 μL of a 3M ammonium sulfate stock solution was thenadded. Coating of the magnetic particles was carried out for 24 hours at37° C. with gentle mixing on a tilt rocker/shaker. After coating, themagnet was applied and the unbound antibody solution removed. Magneticparticles were then washed 2×1 mL for 10 min each at 37° C. with gentlemixing using TE-NaCl buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 2M NaCl)supplemented with 0.1M glycine. Magnetic particles were then rinsed 2×briefly with 1 mL using TE-Saline buffer (10 mM Tris-HCl, pH 8.0, 1 mMEDTA, 200 nM NaCl) supplemented with 0.5% (w/v) BSA and 0.05% (v/v)Tween-20. Magnetic particles were then blocked overnight in 1 mL of thesame buffer at 37° C. with gentle mixing on a tilt rocker/shaker. Afterblocking, magnetic particles were then rinsed 3× briefly in 1 mL usingTE-Saline buffer supplemented with 0.01% Tween-20. Lastly, afterremoving all of the final wash buffer, magnetic particles wereresuspended to 120 μL final volume (100 μg/μL; ˜20% v/v bead suspension)in 5 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 200 mM NaCl, 0.01% (v/v) Tween-20in 50% glycerol for storage at −20° C.

Primer Coating of Beads and Solid-Phase Bridge PCR

Primer attachment to 7 micron diameter non-porous plastic beads wasperformed as described in Example 32 (see Example 30 for actual primersequences). Solid-phase bridge PCR with these beads was also performedas described in Example 32 except that BODIPY-FL-dUTP labeling was notperformed and 2 separate solid-phase bridge PCR reactions were performedusing the plasmid derived template described, containing either humanp53 or GST gene inserts.

Attaching the Capture Antibody and Labeling of the Beads

Following the solid-phase bridge PCR reaction, NeutrAvidin was attachedto the biotin on the beads followed by attachment of the anti-HSV tagPC-antibody as described in Example 31, except that 2.5 μL bead volumewas used per sample and 0.01% Tween-20 (nuclease-free) was included inall buffers (including the NeutrAvidin and PC-antibody solutions) toavoid bead aggregation. Note that the 2 bead species (p53 and GST DNA)were kept separate during these procedures. After loading the anti-HSVtag PC-antibody, the 2 beads species (p53 and GST DNA) were labeled withdifferent fluorophores to enable down streamidentification. To wash andmanipulate the beads or exchange the buffers, 0.45 micron pore size,PVDF membrane, micro-centrifuge Filtration Devices were used unlessotherwise noted (Ultrafree-MC Durapore Micro-centrifuge FiltrationDevices, 400 μL capacity; Millipore, Billerica, Mass.). 1 μL packed beadvolume of each bead species was washed 3×400 μL briefly with 200 mMsodium bicarbonate, 200 mM NaCl and 0.001% (v/v) Tween-20. Beads wererecovered from the aforementioned Filtration Devices in 100 μL of thesame buffer, transferred to 0.5 mL micro-centrifuge tubes, spun downbriefly in a micro-centrifuge at 13,000 rpm and the fluid supernatantremoved. Beads were then resuspended in 10 μL of the same buffer.Fluorescence labeling reagents, either a 25 mM Cy5—NHS monoreactiveester (Amersham Biosciences Corp., Piscataway, N.J.) stock in DMSO or a12.5 mM Alexa Fluor® 488 5-TFP (Invitrogen Corporation, Carlsbad,Calif.) stock in DMF, were freshly diluted to 250 μM in purified waterand 1.6 μL of that was immediately added to the bead suspension. p53 DNAbeads were labeled with Cy5 and GST beads with Alexa Fluor® 488.Reactions were carried out for 15 min with mixing and protected fromlight. After the labeling reaction, each bead suspension was mixed with400 μL of 200 mM sodium bicarbonate, 2 M NaCl, 0.2M glycine, 1 mM EDTAand 0.001% (v/v) Tween-20 to quench the reaction. The beads were thenwashed 1×400 μL briefly with the same buffer followed by 2×400 μLbriefly with TE-Saline buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 200 mMNaCl) supplemented with 0.001% Tween-20.

Cell-Free Protein Expression of the Beads

Cell-free protein expression of the beads was then performed asdescribed in Example 30 with the following exceptions: Beads were onlypre-washed 1× briefly with 400 μL nuclease-free water prior toexpression. 1 μL bead volume was used in a 50 μL cell-free expressionreaction. No tRNA mediated labeling was performed, neither withBODIPY-FL nor PC-biotin or otherwise. The reaction was performed for 1hour with shaking. Since the capture PC-antibody directed against thecommon HSV epitope tag in all expressed proteins is attached to thebeads (see earlier in this Example), in situ protein capture does occurin this case (however, p53 and GST protein expression was performed inseparate tubes).

Probing Expressed Beads with an Anti-p53-Cy5 Antibody and Isolation withMagnetic Particles

Following expression of the solid-phase bridge PCR beads and in situprotein capture, GST and p53 beads (1 μL bead volume each) wereseparately washed 1×400 μL with TBS-T follow by 2×400 μL with 5% BSA(w/v) in TBS-T. To wash and manipulate the beads or exchange thebuffers, 0.45 micron pore size, PVDF membrane, micro-centrifugeFiltration Devices were used unless otherwise noted (Ultrafree-MCDurapore Micro-centrifuge Filtration Devices, 400 μL capacity;Millipore, Billerica, Mass.). Unless otherwise noted, all washes arebrief, 1-3 sec, by vortex mixing. The p53 and GST beads were thensuspended to 500 μL total volume with 5% BSA (w/v) in TBS-T. 250 μL ofthe GST bead suspension was mixed with 2.5 μL of the p53 bead suspensionto form a mixture of approximately 1% p53 beads and 99% GST beads.

The fluid was removed from the p53-GST bead mixture using theaforementioned Filtration Devices and the beads probed with a mousemonoclonal anti-p53-Cy5 fluorescence labeled antibody. The antibody wasthat previously described in Examples 26 and 31 except that the antibodystock was additionally clarified for 1 min in a micro-centrifuge at13,000 rpm to remove particulate prior to use. The clarified antibodystock was then diluted 1/20 with 5% BSA (w/v) in TBS-T and the dilutedantibody again clarified for 1 min in a micro-centrifuge followed bypassing the supernatant through the aforementioned Filtration Devices.The filtrate, corresponding to the diluted and clarified antibody, wasthen used to probe the bead mixture. 100 μL was used to probe the beadmixture for 30 min at room temperature with gentle mixing. The beadmixture was washed 3×400 μL briefly with TBS-T, then resuspended with100 μL of 5% BSA (w/v) in TBS-T and transferred out of the FiltrationDevice into a 0.5 mL micro-centrifuge tube.

50 μL of the resultant bead suspension was used for imaging of theun-separated beads and the remaining 50 μL was set aside for magneticparticle isolation (see later in this Example). For imaging theun-separated beads, the beads were spun down briefly in amicro-centrifuge at 13,000 rpm and the supernatant completely removed.The bead pellet was resuspended in 5 μL of acrylamide mix and the entirepopulation was embedded in a thin polyacrylamide film on top of a glassmicroscope slide under an 18 mm round cover glass, as described inExamples 32 and 34. The embedded beads were fluorescently imaged (seelater in this Example).

The remaining 50 μL of un-separated bead mixture (i.e. that which wasnot embedded) was then used to test selective purification of the beadpopulation bearing the p53 protein and hence the bound mouseanti-p53-Cy5 antibody. This was achieved using 1 micron magneticparticles that were coated with an anti-[mouse IgG] species specificsecondary antibody; which selectively bind the mouse anti-p53-Cy5antibody but not the rabbit anti-HSV PC-antibody also on the beads.First, 1 μL (100 μg) of the secondary antibody coated magnetic particles(prepared as described earlier in this Example) was pre-washed 1×400 μLbriefly with 5% BSA (w/v) in TBS-T. All washes involving the magneticparticles were performed in 0.5 mL polypropylene micro-centrifuge tubesand using the magnet system described earlier in this Example, unlessotherwise noted. After removing the wash solution, the pelletcorresponding to the magnetic particles was gently resuspended with the50 μL of un-separated p53-GST plastic bead mixture. To allow themagnetic particles to bind the targeted p53 beads, the mixture wasallowed to stand for 30 min with gentle intermittent mixing (˜every 5min). Mixing was performed by manually pipetting the suspensionup-and-down. The magnetic particles and any beads bound to them werewashed 3×400 μL briefly with TBS-T. Washes were performed by gentlyresuspending the bead and magnetic particle mixture by manuallypipetting up-and-down, applying the aforementioned magnet for ˜15 sec todraw the magnetic particles and any bound beads to the side-wall of thetube, and then gently removing the fluid containing the suspended 7micron diameter plastic beads that were not bound to magnetic particles(while magnetic particles remain adherent to the side-walls of thetube). After washing, the magnetic particles and bound beads were spundown briefly in a micro-centrifuge and the supernatant completelyremoved. The bead pellet was resuspended in 5 μL of acrylamide mix andthe entire population was embedded in a thin polyacrylamide film forimaging as described earlier in this Example for the un-separated beads.

Un-separated and purified beads, embedded in a polyacrylamide film, wereimaged for fluorescence using the ArrayWoRx^(e) BioChip fluorescencereader (Applied Precision, LLC, Issaquah, Wash.).

Results:

Representative regions of the fluorescence bead images (embedded beads)are shown in FIG. 30. The un-separated 1% p53 and 99% GST bead mix (leftpanels FIG. 30) and the purified p53 beads (right panels FIG. 30) wereimaged in both the green and red fluorescence channels, corresponding toGST beads (labeled with Alexa Fluor® 488) and p53 beads (labeled withCy5), respectively (upper and lower panels of FIG. 30 respectively). Thegreen fluorescence channel for the un-separated beads is shown in theupper left panel of FIG. 30, corresponding to GST beads. The same regionwas also imaged in the red fluorescence channel (lower left panel FIG.30), corresponding to the p53 beads. Although representative regions areshown in FIG. 30, the entire bead populations were enumerated (142,127total un-separated beads and 1,193 total beads following purification),and the actual measured percentage of p53 beads in the un-separatedmixture was indeed 1%. The contaminating green beads (GST) followingpurification with the magnetic particles are shown in the upper rightpanel of FIG. 30, while imaging of the same region in the redfluorescence channel (lower right panel FIG. 30) shows the purified redbeads (p53). Enumeration of the entire bead population in the purifiedsample shows that the targeted red beads (p53) are 52.7% pure, thuscorresponding to a more than 50-fold enrichment factor and removal of99.6% of the contaminating green beads (GST) using the magnetic particlepurification technique. The yield of targeted red (p53) beads was 42.4%of the starting number of un-separated red (p53) beads.

Example 34 Contact Photo-Transfer of Peptides onto Solid Surfaces usedfor Downstream MALDI-TOF Mass Spectrometry Analysis Preparation of theAnti-Flag Pc-Antibody Affinity Resin

A mouse monoclonal anti-FLAG tag antibody clone M2 was purchasedcommercially (Sigma-Aldrich, St. Louis, Mo.). 242 μL of the antibodysolution as provided by the manufacturer (4.9 μg/μL) was desalted on aNAP-10 column according to the manufacturer's instructions (AmershamBiosciences Corp., Piscataway, N.J.) against a 200 mM sodium bicarbonateand 200 mM NaCl buffer. The antibody was then labeled by adding an AlexaFluor® 488 5-TFP labeling reagent (Invitrogen Corporation, Carlsbad,Calif.) at a 2-fold molar excess from a 12.5 mM stock in DMF. Thereaction was carried out for 30 min with gentle mixing. Next, a 20-foldmolar excess (relative to antibody) of AmberGen's PC-biotin-NHS labelingreagent was added to the reaction from a 50 mM stock in DMF. Thereaction was carried out for an additional 30 min with gentle mixing. 1mL of the Alexa Fluo® 488 and PC-biotin dual labeled PC-antibody wasthen separated from the un-reacted labeling reagent using a NAP-10column according to the manufacturer's instructions (AmershamBiosciences Corp., Piscataway, N.J.) against TBS. The resultantPC-antibody solution (0.42 μg/μL) was supplemented to 0.1% (w/v) withBSA from a 10% stock in water. 750 μL (˜300 μg) of this solution wasthen added to 300 μL packed bead volume of NeutrAvidin agarose beads(Pierce Biotechnology, Inc., Rockford, Ill.) which were pre-washed 4×1mL briefly with 0.1% (w/v) BSA in TBS. PC-antibody capture was carriedout for 30 min with gentle mixing. The beads were then washed 4×5 mineach with 1 mL of 0.1% (w/v) BSA in TBS. Beads were then washed 3×1 mLbriefly with TBS followed by 2×1 mL briefly with 50% TBS, 50% glyceroland 1.5 mM sodium azide and the beads resuspended to a 30% (v/v)suspension in the same buffer for storage at −20° C. Based onmeasurements of the fluorescence Alexa Fluor® 488 label on thePC-antibody, 82% of the PC-antibody was captured on the beads for 0.8 μgof PC-antibody per 1 μL of packed beads.

Cell-Free Expression of BRCA Peptides and Affinity Capture

Isolation of genomic DNA from cultured cells (HeLa cells; ATCC;Manassas, Va.) and PCR amplification of fragments of the human BRCA2gene was performed essentially as reported by AmberGen in the scientificliterature for the human APC gene [Gite et al. (2003) Nat Biotechnol 21,194-197], except that an N-terminal FLAG epitope tag (amino acidsequence DYKDDDDK [SEQ NO. 14]) was the only epitope tag incorporatedinto the expressed sequences. Epitope tags and elements necessary forefficient cell-free expression were introduced into the PCR amplicon byway of specialized primers [Gite et al. (2003) Nat Biotechnol 21,194-197]. PCR primers for 2 gene fragments, designated CT64 and CT61, ofthe BRCA2 gene, were as follows:

Forward CT64: [SEQ NO. 15]5′TAATACgACTCACTATAgggAgAggAggTATATCAATggATTATAAAgACgATgATgATAAAAgTACAgCAAgTggAAAgCAA3′ Reverse CT64: [SEQ NO. 16]5′TTATTTATTTATTTTTgATACATTTTgTCTAgA3′ Forward CT61: [SEQ NO. 17]5′TAATACgACTCACTATAgggAgAggAggTATATCAATggATTATAAAgACgATgATgATAAACTTCATAAgTCAgTCTCATCT3′ Reverse CT61: [SEQ NO. 18]5′TTATTTATTTATTTCTATTTCAgAAAACACTTg3′

Cell-Free protein expression of the crude PCR amplicon was performed inthe E. coli based PureSystem (Post Genome Institute Co., LTD., Japan)according to the manufacturer's instructions (40 μL expression persample reacted for 1 hour at 42° C.). A negative control expressionreaction (−DNA), lacking only the necessary expressible PCR DNA was alsoperformed. Following cell-free expression, reactions were mixed withequal volume of 2× concentrated PBS with 0.2% (v/v) Triton X-100.Samples were mixed gently for 5 min at +4° C. Samples were clarified at13,000 rpm in a micro-centrifuge for 1 min and the supernatantcollected.

To capture the cell-free expressed FLAG epitope tagged peptides from thecrude reaction, 5 μL packed bead volume of the aforementioned preparedanti-FLAG PC-antibody beads was pre-washed 2×400 μL briefly with 0.1%(v/v) Triton X-100 in PBS. The aforementioned processed expressionsamples were then added to the washed bead pellets and mixed for 30 minat +4° C. to allow peptide capture on the beads. Beads were then washed2×400 μL briefly with PBS then 1×400 μL briefly with 50% glycerol inPBS. For subsequent use in contact photo-transfer, beads were thenadjusted to a 50% (v/v) suspension (slurry) in the 50% glycerol and PBSbuffer.

Contact Photo-Transfer of Captured BRCA Peptides and Mass Spectrometry

To demonstrate the compatibility of contact photo-transfer withmatrix-assisted laser desorption/ionization time of flight (MALDI-TOF)mass spectrometry, a 0.5 μL droplet (˜2 mm diameter) of theaforementioned 50% (v/v) bead slurry was applied to the surface of anepoxy activated glass microarray slide (SuperEpoxy substrates, TeleChemInternational, Inc. ArrayIt™ Division, Sunnyvale, Calif.). Droplets wereapplied in defined areas outlined with black magic marker to allow lateridentification. To photo-transfer the antibody and any bound peptidefrom the beads to the microarray slide (contact photo-transfer), theslide was illuminated from above with near-UV light for 5 min (365 nmpeak lamp; Blak-Ray Lamp XX-15, UVP, Upland, Calif.; used at 1 to 3mW/cm²). After allowing binding of the photo-released material to theepoxy activated slide by incubating for 30 min at 37° C. in a humidifiedchamber, beads were gently washed away by rinsing 2× briefly in excess0.1M glycine in TBS followed by 2× briefly in purified water, in a traywith shaking. Bead removal was verified by visible microscopy.Microarray slides were dried by centrifugation in a padded tube and,prior to MALDI-TOF, the slide was imaged for fluorescence in theArrayWoRx^(e) BioChip reader (Applied Precision, LLC, Issaquah, Wash.).For MALDI-TOF, a saturated matrix solution was prepared by dissolving 25mg of α-cyano-4-hydroxycinnamic acid in 1250 μL of 50% (v/v)acetonitrile and 0.3% (v/v) trifluoroacetic acid. The solution was mixedvigorously for 10 min and clarified at 13,000 rpm in a micro-centrifuge.The supernatant was collected and used as the matrix solution. The spotswere then overlaid with 0.2 μL of matrix solution which was then allowedto crystallize. Next, the microarray slide was cut and mounted onto acustom designed frame for insertion into the MALDI-TOF instrument(Voyager-DE; Applied Biosystems; Foster City, Calif.). Importantly,MALDI-TOF from glass slides, without the use of contact photo-transfer,has been previously published [Mehlmann et al. (2005) Anal Bioanal Chem382, 1942-1948]. In this Example, MALDI-TOF was performed with thefollowing instrument parameters: Instrument mode linear; positive ionmode; delayed extraction mode at 180 nsec; accelerating voltage 25,000;grid voltage 90.000; guide wire voltage 0.100; and a laser intensitysetting of 2,800.

Results:

As shown in FIG. 31A, fluorescence imaging of the microarray slide priorto MALDI-TOF verified successful photo-transfer of the Alexa Fluor® 488labeled anti-FLAG PC-antibody in all cases. FIG. 31B shows the resultsof MALDI-TOF on the contact photo-transfer fabricated microarray slides.The minus DNA negative control sample (−DNA) shows no measurable peaks,while the CT61 and CT64 peptides are observed at essentially the correctmass positions (±1%). Other embodiments of this Example are possiblewhere contact photo-transfer is performed onto activated or coated metalMALDI plates or targets, instead of onto activated or coated glassmicroarray slides. For example, contact photo-transfer will be performedonto activated (chemically reactive), secondary antibody coated (tocapture photo-released PC-antibody) or polymer coated metal MALDIplates, which is expected to improve signal-to-noise ratios, peakresolution and mass accuracy. Gold and other metal plates compatiblewith MALDI-TOF have been reported with various coatings or activationsincluding amine-reactive moieties to attach proteins [Neubert et al.(2002) Anal Chem 74, 3677-3683], charged or hydrophobic protein bindingpolymers such as poly-lysine or nitrocellulose [Jacobs & Dahlman. (2001)Anal Chem 73, 405-410; Zhang & Orlando. (1999) Anal Chem 71, 4753-4757]and even biotin coatings which have been used for creating proteinmicroarrays for MALDI-TOF readout [Koopmann & Blackburn. (2003) RapidCommun Mass Spectrom 17, 455-462].

Example 35 Contact Photo-Transfer of DNA: Hybridization ProbingPreparing PC-Biotin Labeled DNA and Loading to Beads

A 5′ C6 (6-carbon spacer) amine modified oligonucleotide was purchasedfrom Sigma-Genosys (The Woodlands, Tex.) having the following sequence:

5′[Amine]gTTAAATTgCTAACgCAgTCAggAg3′ [SEQ NO. 19]

The oligonucleotide was prepared to a 10 μg/μL stock in nuclease-freewater and clarified in a micro-centrifuge for 1 min at 13,000 rpm. 100μL of the supernatant was then passed through a NAP-5 desalting columnaccording to the manufacturer's instructions (Amersham BiosciencesCorp., Piscataway, N.J.) against a 200 mM sodium bicarbonate and 200 mMNaCl buffer (nuclease-free reagents). 400 μL of the resultant 1 μg/μLoligonucleotide was then labeled with a 20-fold molar excess ofAmberGen's PC-biotin-NHS labeling reagent (added from a 50 mM stock inanhydrous DMF). The reaction was carried out for 30 min with gentlemixing. As a negative control, an equal amount of oligonucleotide wasnot labeled, but was otherwise processed in parallel in the same manner.Each sample was then passed through a NAP-5 desalting column accordingto the manufacturer's instructions (Amersham Biosciences Corp.,Piscataway, N.J.) against TE-NaCl buffer (10 mM Tris-HCl, pH 8.0, 1 mMEDTA, 2M NaCl). The resultant oligonucleotides were 0.4-0.5 μg/μL.

To load the oligonucleotides onto beads, 25 μL packed bead volume ofNeutrAvidin agarose beads (Pierce Biotechnology, Inc., Rockford, Ill.)was washed 3×400 μL with TE-NaCl buffer. To wash the beads or exchangethe buffers, 0.45 micron pore size, PVDF membrane, micro-centrifugeFiltration Devices were used unless otherwise noted (Ultrafree-MCDurapore Micro-centrifuge Filtration Devices, 400 μL capacity;Millipore, Billerica, Mass.). 400-500 μL of the aforementioned preparedoligonucleotide solutions (PC-biotin labeled or unlabeled DNA) was thenused to resuspend the washed bead pellets and bead capture of theoligonucleotides was allowed to occur for 30 min with gentle mixing.Beads were then washed 2×400 μL briefly with TE-NaCl and then 2×400 μLbriefly with 1 mM EDTA in PBS. For final washing and bead storage,either PBS, 1 mM EDTA and 50% glycerol or 50 mM sodium phosphate, pH7.5, 2M NaCl and 50% glycerol was used with similar results. Finalwashing was 2×400 μL briefly and beads were stored at −20° C. as 10-20%(v/v) suspensions.

For quality control purposes, 1 μL packed bead volume of the beadsloaded with the PC-biotin labeled oligonucleotide or the unlabeledoligonucleotide were stained with the OliGreen ssDNA detection reagentas described in Example 30. To additionally verify the boundoligonucleotide, 5 μL packed bead volume was washed 3×400 μL brieflywith 6×SSPE buffer (50 mM sodium phosphate, pH 7.5, 900 mM NaCl and 6 mMEDTA). The beads were then resuspended in 50 μL of a complementaryoligonucleotide probe (10 μM in 6×SSPE) labeled on its 5′ end with theCy5 fluorophore (Sigma-Genosys; The Woodlands, Tex.) and having thefollowing sequence:

5′[Cy5]CTCCTgACTgCgTTAgCAATTTAAC3′ [SEQ NO. 20]

The probe was allowed to hybridize with the beads for 30 min at 42° C.with gentle mixing. Beads were then washed 2×400 μL briefly with 6×SSPE,2×400 μL briefly with 3×SSPE, 1×400 μL briefly with 50 mM sodiumphosphate, pH 7.5, 2M NaCl and 50% glycerol and lastly resuspended in500 μL of the same buffer. 250 μL of bead suspension (2.5 μL packedbeads) was placed in a thin-walled polypropylene 0.5 mL PCR tube and thebeads were spun down briefly in a micro-centrifuge at 13,000 rpm. Mostof the supernatant was removed (except ˜10 μL) and the bead pellet wasimaged with a FUJIFILM FLA-2000 fluorescence scanner (Fuji Photo FilmCo., LTD., Equipment Product Division, Science Group, Japan) directly inthe tubes, using the 633 nm He—Ne laser and a 675 nm emissions filter.

Preparation of a NeutrAvidin-Cy5 Labeled Conjugate

For indirect fluorescence detection of contact photo-transfer fabricatedDNA microarrays that are hybridized with biotinylated complementaryoligonucleotide probes, as described later in this Example, aNeutrAvidin-Cy5 labeled fluorescent conjugate was first prepared asdescribed in this paragraph. NeutrAvidin powder (Pierce Biotechnology,Inc., Rockford, Ill.) was dissolved to 5 mg/mL in purified water andthen 350 μL was passed through a NAP-5 desalting column according to themanufacturer's instructions (Amersham Biosciences Corp., Piscataway,N.J.) against a 200 mM sodium bicarbonate and 200 mM NaCl buffer. Theresultant 1 mg/mL NeutrAvidin solution was labeled using 10 molarequivalents of a Cy5—NHS monoreactive ester (Amersham Biosciences Corp.,Piscataway, N.J.) that was added from a 27 mM stock in DMSO. Thereaction was carried out for 30 min with gentle mixing. The conjugatewas then passed through a NAP-10 desalting column according to themanufacturer's instructions (Amersham Biosciences Corp., Piscataway,N.J.) against TBS to remove un-reacted labeling reagent. Measurement ofabsorbance at 280 nm and 649 nm determined that there was approximately1 Cy5 molecule per protein molecule on average. The NeutrAvidin-Cy5conjugate solution was then diluted 1:1 with 100% glycerol for storageat −20° C. (0.38 μg/μL after dilution).

Contact Photo-Transfer of DNA and Detection by Hybridization Probing andIndirect Fluorescence

For contact photo-transfer, only the aforementioned beads that wereloaded with the PC-biotin labeled DNA were used (i.e. not beads thatwere treated with the unlabeled DNA). Furthermore, the beads used werethose remaining beads that were not stained with OliGreen and notpreviously probed with the Cy5 labeled complementary oligonucleotide, asdescribed earlier in this Example for quality control purposes. To washthe beads or exchange the buffers, 0.45 micron pore size, PVDF membrane,micro-centrifuge Filtration Devices were used unless otherwise noted(Ultrafree-MC Durapore Micro-centrifuge Filtration Devices, 400 μLcapacity; Millipore, Billerica, Mass.). 5 μL packed bead volume wastaken and washed 1×400 μL briefly with 6×SSPE then 1×400 μL for 15-45min at 42° C. with gentle mixing. Next, beads were washed 3×400 μLbriefly with nuclease-free water. Beads were then washed 1×400 μL for 15min at 42° C. using 50 mM sodium phosphate, pH 7.5, 2M NaCl and 50%glycerol with gentle mixing and then rinsed 1×400 μL briefly in the samebuffer. Beads were then resuspended to 1-2% beads (v/v) in 50 mM sodiumphosphate, pH 7.5, 2M NaCl and 50% glycerol for use in contactphoto-transfer.

For contact photo-transfer, 45-50 μL of the aforementioned beadsuspension was applied to an epoxy activated microarray slide(SuperEpoxy substrates, TeleChem International, Inc. ArrayIt™ Division,Sunnyvale, Calif.) and overlaid with a standard 18 mm round microscopecover glass. The slide was allowed to stand 5 min undisturbed to allowthe beads to settle to the slide surface. Without further disturbance,the slide was then illuminated from above with near-UV light for 5 min(365 nm peak lamp; Blak-Ray Lamp XX-15, UVP, Upland, Calif.; used at 1to 3 mW/cm²). A minus light negative control was performed on the sameslide, by masking a region of the slide with a black plastic opaque lidthat was lined with aluminum foil. After light treatment, binding of thephoto-released material to the epoxy activated slide was allowed tooccur by incubating for 20 min at room temperature in a humidifiedchamber without disturbance. Beads and cover glasses were then removedand the slides simultaneously washed/blocked by treating 2×15 min withexcess 10 mM Tris-HCl, pH 8.0, 1 mM EDTA and 900 mM NaCl at 42° C. in atray with shaking. Slides were further blocked for 5 min at 42° C. withexcess 0.1% (w/v) nuclease-free BSA in 10 mM Tris-HCl, pH 8.0, 1 mM EDTAand 900 mM NaCl. Slides were then rinsed 3× briefly with excessnuclease-free water and dried by centrifugation in a padded tube.

Dried slides were then probed with a complementary oligonucleotidefollowed by detection by indirect fluorescence. For indirectfluorescence, slides were probed with a complementary oligonucleotidethat was 5′ labeled with biotin (Sigma-Genosys; The Woodlands, Tex.) andhaving the following sequence:

5′[Biotin]CTCCTgACTgCgTTAgCAATTTAAC3′ [SEQ NO. 21]

The probing solution was comprised of 10 μM of the biotin labeledcomplementary oligonucleotide and 10 mM d-biotin in 6×SSPE buffer. Freed-biotin was included as a precautionary measure to prevent binding ofthe probe to any NeutrAvidin that may have leached from the beads andbound to the microarray slide. Probing the microarray slide was achievedusing 120 μL of the solution under a standard 22×60 mm microscope coverglass overlay, for overnight at 42° C. in a humidified chamber. Slideswere then allowed to cool to room temperature for 30 min followed bywashing with excess 6×SSPE in a tray with mixing for 3×1 min each.Slides were then treated with 100 μL of the aforementioned preparedNeutrAvidin-Cy5 conjugate diluted to 3.8 μg/mL in 6×SSPE supplementedwith 1% (w/v) nuclease-free BSA. Treatment was performed under astandard 22×60 mm microscope cover glass overlay, for 30 min at 37° C.in a humidified chamber. Sides were then washed (cover glass removed)with excess 6×SSPE in a tray with mixing for 3×1 min each. Slides werethen dried by centrifugation in a padded tube and imaged forfluorescence (see later in this Example).

Contact Photo-Transfer of DNA and Detection by Hybridization Probing andDirect Fluorescence

Direct fluorescence probing of the microarray slides containing the DNAspots was performed essentially the same as with the indirectfluorescence method described above in this Example. After performingcontact photo-transfer as described above in this Example, binding ofthe photo-released material to the epoxy activated slide was allowed tooccur by incubating for 20 min at room temperature in a humidifiedchamber without disturbance. Beads and cover glasses were then removedand the slides simultaneously washed/blocked by treating 2×15 min withexcess 10 mM Tris-HCl, pH 8.0, 1 mM EDTA and 900 mM NaCl at 42° C. in atray with shaking. Slides were further blocked for 10 min at 42° C. withexcess 0.1% (w/v) nuclease-free BSA in 10 mM Tris-HCl, pH 8.0, 1 mM EDTAand 900 mM NaCl. Slides were then rinsed 3× briefly with excessnuclease-free water and dried by centrifugation in a padded tube.

Dried slides were then probed with a complementary oligonucleotide whichwas directly labeled with fluorescence. The oligonucleotide probe was 5′labeled with Cy5 (Sigma-Genosys; The Woodlands, Tex.) and having thefollowing sequence:

5′[Cy5]CTCCTgACTgCgTTAgCAATTTAAC3′ [SEQ NO. 22]

The probing solution was comprised of 10 μM of the Cy5 labeledcomplementary oligonucleotide in 6×SSPE buffer. Probing the microarrayslide was achieved using 100 μL of the solution under a standard 22×60mm microscope cover glass overlay, for 15 min at 42° C. in a humidifiedchamber. Slides were then allowed to cool to room temperature for 15 minfollowed by washing with excess 6×SSPE in a tray with mixing for 4×1 mineach. Slides were then dried by centrifugation in a padded tube andimaged for fluorescence (see later in this Example).

Imaging of Contact Photo-Transfer Fabricated DNA Microarrays

Imaging of fluorescent signals from the Cy5 probes was achieved using anArrayWoRx^(e) BioChip fluorescence reader (Applied Precision, LLC,Issaquah, Wash.) with the appropriate standard built-in filter set.

Results:

Results in FIG. 32A show verification of DNA attachment to the agarosebeads prior to use in contact photo-transfer. Beads that were verifiedwere those that were loaded with either the PC-biotin labeled DNA (+PCB)or beads that were treated with an equivalent amount of unlabeled DNA(−PCB), as a negative control for non-specific binding. The upper panelsshow detection with the ssDNA fluorescent stain OliGreen and the lowerpanels show detection with a directly Cy5 labeled complementaryoligonucleotide probe. After detection, the bead pellets were imageddirectly in thin-walled 0.5 mL polypropylene micro-centrifuge tubes. Inboth cases, bound DNA is specifically detected only on beads loaded withthe PC-biotin labeled DNA (+PCB).

FIG. 32B shows the DNA spots applied to the microarray slide via contactphoto-transfer and detected with either a biotin labeled complementaryoligonucleotide probe followed by NeutrAvidin-Cy5 detection upper leftand right panels) or DNA spots that were detected with a directlylabeled Cy5 fluorescent complementary oligonucleotide probe (lower leftand right panels). In either case, a light-dependent transfer of the DNAfrom the beads to the microarray slide is shown, forming discretemicroarray spots (upper and lower right panels). In the case of indirectfluorescence, no detectible spots are visible when contactphoto-transfer was performed in the absence of proper light illumination(upper left panel). In the case of direct fluorescence detection, tracesignal is observed when contact photo-transfer was performed in theabsence of light illumination (lower left panel), likely due to thehigher sensitivity of this method. This signal presumably pertains toDNA leaching off the beads during the contact photo-transfer process andquantification shows the signal to be only 18% (˜5-fold less) of thetotal signal achieved when proper light illumination is used for contactphoto-transfer (lower right panel).

Example 36 Effective Single Template Molecule Solid-Phase Bridge PCR:Amplicon Detection Through Fluorescence dUTP Labeling During the PCRReaction

Examples 36-39 demonstrate a step-wise process by which to verify, usingDNA level assays, that only one or a few of the original templatemolecules are amplified per bead during solid-phase bridge PCR. One keyparameter is the proper concentration of template initially added to theprimer coated beads to achieve this result, and this, referred to as the“target template concentration”, will vary depending on thecharacteristics of a given solid-phase bridge PCR system. Thesecharacteristics effect the efficiency of initially capturing thetemplate onto the beads and/or the efficiency of template amplification.For example, characteristics of the template and primer pair combinationsuch as template length, sequence-dependent secondary structure of thetemplate and/or primer and primer melting temperature (T_(m)).Characteristics of the beads such as bead composition (e.g. polar,charged or hydrophobic material) and porosity (e.g. whether pores arepresent and pore size) as well as primer density, will also effect thetarget template concentration. Lastly, characteristics of thesolid-phase bridge PCR reaction itself impact the target templateconcentration, such as annealing temperatures used and additives such assalt or dimethyl sulfoxide, which effect the primer and template meltingtemperatures (T_(m)). Therefore, this target template concentration willvary and needs to be systemically determined for any given solid-phasebridge PCR system using the generalized approach detailed in Examples36-39.

The generalized approach uses a binary system, whereby 2 distincttemplate DNA species, flanked by common sequences at the 5′ and 3′ endsto which the primers are directed, are initially added to the primercoated beads for solid-phase bridge PCR amplification. The first stageinvolves narrowing down the target template concentration usingdetection of the DNA amplicon (solid-phase bridge PCR amplificationproduct) on individual beads, generically, using incorporation of afluorescently labeled deoxynucleotide triphosphate during thesolid-phase bridge PCR reaction itself (this Example 36 and thesubsequent Example 37). The target template concentration is thenconfirmed by detecting and distinguishing both amplicon species onindividual beads using dual fluorescence hybridization probing. Theexpected result is that individual beads should contain ampliconcorresponding to primarily one (e.g. >70%, still more preferably greaterthan 80%, and preferably 90% or more), but not both of the template DNAspecies (Example 38). Lastly, the target template concentration isvalidated by titrating the ratio of the 2 template species initiallyadded to the beads. The expected result after solid-phase bridge PCRamplification is that the ratio of individual beads containing eachamplicon should approximately (plus or minus 20%, more preferably, plusor minus 10% or less) reflect the ratio of template species initiallyadded to the beads (Example 39).

Preparing the Solid-Phase Bridge PCR Template DNA:

Note: All buffers and reagents used throughout this entire Example,unless otherwise noted, were minimally DNAse, RNAse and protease free,referred to as Molecular Biology Grade (MBG), including the water,referred to as MBG-Water.

Full length human p53 (GeneBank NM_(—)000546) and GST A2 (GeneBankNM_(—)000846) genes (open reading frame) were cloned into the pETBlue-2plasmid (EMD Biosciences, Inc., San Diego, Calif.) according to standardpractices and the manufacturer's instructions. Plasmids were then usedas template for standard solution-phase PCR with gene-specific primers,using standard molecular biology practices. The primers are listed belowwhereby the bracketed sequences indicate the gene-specific hybridizationregions, while the remaining sequences are non-hybridizing regions whichact as common universal sequences, flanking the gene inserts, to whichthe subsequent solid-phase bridge PCR primers are directed (thenon-hybridizing regions also correspond to elements needed for latercell-free protein expression as well as epitope tag detection):

p53 Forward Primer: [SEQ NO. 23]5′ggATCCTAATACgACTCACTATAgggAgAggAggTATATCAATggATTATAAAgACgATgATgATAAA[gAggAgCCgCAgTCAgATCCTAgCgT C]3′ p53 Reverse Primer:[SEQ NO. 24] 5′TTTTTATTACTTACCCAggCggTTCATTTCgATATCAgTgTATTTATTTTAT[CAAgggggACAgAACgTTgTTTTCA]3′ GST A2 Forward Primer: [SEQ NO. 25]5′ggATCCTAATACgACTCACTATAgggAgAggAggTATATCAATggATTATAAAgACgATgATgATAAA[gCAgAgAAgCCCAAgCTCCACTACTC C]3′ GST A2 ReversePrimer: [SEQ NO. 26] 5′TTTTTATTACTTACCCAggCggTTCATTTCgATATCAgTgTATTTATTTAT[CTCTTCAAACTCTACTCCAgCTgCAgCC]3′Following the solution-phase PCR, the products were analyzed by standardagarose gel electrophoresis and ethidium bromide staining to ensure asingle band was produced and of the correct molecular weight. Based onthe primers used, gene fragments of human p53 and human GST A2, flankedby common universal sequences, are produced as the PCR product, at 221and 212 bp respectively. The PCR products were then purified by agarosegel electrophoresis and the resultant DNA concentration was 83-84 ng/μL.From here forward, these purified PCR products are referred to as“Concentrated Stock Template DNA Solutions”, and were subsequently usedto make the template DNA dilutions for the solid-phase bridge PCRreactions described later in this Example.

Preparation of Agarose Beads Covalently Conjugated to PCR Primers Usedfor Solid-Phase Bridge PCR:

The following universal forward and reverse PCR primers, directedagainst the common sequences in both the human p53 and human GST A2 DNAtemplates (templates prepared as described earlier in this Example),were purchased from Sigma-Genosys (The Woodlands, Tex.), both with a 5′primary amine modification following a 6 carbon spacer:

[SEQ NO. 27] Forward: 5′[Amine]TAATACgACTCACTATAgggAgAggAgg3′ [SEQ NO.28] Reverse: 5′[Amine]TTACTTACCCAggCggTTCATTTC3′

Primary amine reactive, NHS ester activated (N-hydroxysuccinimide), 4%cross-linked agarose beads (˜100 micron diameter) were purchased fromAmersham Biosciences (Amersham Biosciences Corp., Piscataway, N.J.). Thefollowing procedures, unless otherwise noted, were performed in batchmode using Filtration Devices to facilitate manipulation of the beadedmatrix (˜100 micron beads), perform washes and otherwise exchange thebuffers (Filtration Devices=Ultrafree-MC Durapore Micro-centrifugeFiltration Devices, 400 μL capacity, PVDF filtration membrane, 0.45micron pore size; Millipore, Billerica, Mass. distributed bySigma-Aldrich, St. Louis, Mo.). 200 μL of bead volume (400 μL of stock50% slurry as supplied by the manufacturer) was placed in a FiltrationDevice, spun down briefly in a micro-centrifuge (just until reachesmaximum speed of ˜13,000 rpm corresponding to ˜16,000×g) and thefiltrate corresponding to the isopropanol storage buffer was discarded.The 200 μL of beads was then washed 4× briefly (briefly=5 sec vortexmix) with 400 μL each of ice cold 1 mM HCl prepared in MBG-Water. Unlessotherwise stated, all buffers or washes in this procedure were removedfrom the beads (exchanged) by spinning the Filtration Devices briefly ina standard micro-centrifuge (just until reaches maximum speed of ˜13,000rpm corresponding to ˜16,000×g) and discarding the filtrate. The washedbead pellet was then resuspend in 200 μL containing 125 μM of eachprimer (forward and reverse primers; both primers added from 1,250 μMstocks in MBG-Water) prepared in Binding Buffer (200 mM sodiumbicarbonate, 2M NaCl) additionally containing 12 μM of a Biotin-AmineLinker (EZ-Link Amine-PEO3-Biotin; Pierce Biotechnology, Inc., Rockford,Ill.; added from a 40× concentrated stock in MBG-Water). As a negativecontrol, a second set of beads received the same 200 μL of solutionlacking only the forward and reverse primers. The binding reaction wasallowed to proceed for 1 hour with gentle vortex mixing. The beads werethen washed 1× briefly with 400 μL of Quenching Buffer (200 mM sodiumbicarbonate, 200 mM glycine, 1 mM EDTA, 2M NaCl) and then 2×400 μL withQuenching Buffer for 30 min each with gentle vortex mixing. The beadswere then washed 2× briefly with Binding Buffer followed by 2× for 5 mineach with TE-NaCl (10 mM Tris, pH 8.0, 2M NaCl and 1 mM EDTA). Beadswere lastly washed 1× briefly with SP-PCR Storage Buffer (50% glycerol,10 mM Tris, pH 8.0, 2M NaCl, 1 mM EDTA) and then diluted to a 20% (v/v)bead suspension in SP-PCR Storage Buffer. The bead suspension wasrecovered from the upper chamber of the Filtration Device and stored ina 1.5 mL polypropylene micro-centrifuge tube at −20° C. From hereforward, these beads are referred to as Primer-Conjugated Agarose Beads.

Qualitative Analysis of Primer Attachment:

To qualitatively verify successful primer attachment to thePrimer-Conjugated Agarose Beads, an aliquot of the beads was stainedwith the single-stranded DNA fluorescence-based detection reagentOliGreen (Invitrogen Corporation, Carlsbad, Calif.). The manufacturersupplied reagent was diluted 1/200 in TE (10 mM Tris, pH 8.0, 1 mM EDTA)containing 0.01% (v/v) Tween-20. 5 μL of the prepared Primer-ConjugatedAgarose Bead suspension (20% beads for 1 μL actual bead volume) wasmixed with 100 μL of the diluted OliGreen reagent in a thin-walled 0.5mL clear polypropylene PCR tube. As a negative control, the beads thatwere prepared in the same manner, except lacked any attached primer,were also tested. After approximately 1 min, the beads were spun downbriefly in a micro-centrifuge (just until reaches maximum speed of˜13,000 rpm corresponding to ˜16,000×g), 90 μL of the fluid supernatantwas then removed and the bead pellet imaged directly in the tubes usinga laser-based fluorescence scanner (FUJI FLA-2000, 473 nm solid-statelaser excitation and 520 nm emissions filter) (FUJI Photo Film Co. Ltd,Japan).

First Round of Effective Single Template Molecule Solid-Phase BridgePCR.

5 μL actual bead volume of the previously prepared Primer-ConjugatedAgarose Beads was used per each sample, but first, enough beads for all3 sample permutations were washed in bulk, with heating. To do so, 75 μLof the aforementioned 20% (v/v) Primer-Conjugated Agarose Beadsuspension (15 μL actual bead volume) was placed into a 0.5 mLpolypropylene thin-wall PCR tube. The beads were spun down briefly in astandard micro-centrifuge (just until reaches maximum speed of ˜13,000rpm corresponding to ˜16,000×g). As much of the fluid supernatant wasremoved as possible by manual pipetting, with the beads nearly going todryness. 60 μL of TE-50 mM NaCl (10 mM Tris, pH 8.0, 1 mM EDTA, 50 mMNaCl) was added to the pellet, to bring the volume back to the original20% beads (v/v). The beads were briefly vortex mixed then spun down andall fluid removed as described before. 60 μL of TE-50 mM NaCl was againadded to the pellet as above and the tube placed in a PCR machine(Mastercycler Personal; Eppendorf AG, Hamburg, Germany) at 95° C. for 10min (lid temperature 105° C. and no mineral oil used) (beads wereresuspended by brief gentle vortex mixing just before and at 5 min ofthis step). After heating, the tube was immediately removed from the PCRmachine, the beads diluted in 400 μL of TE-50 mM NaCl and the beadsuspension then transferred to a Filtration Device. Filtration wasperformed and the filtrate discarded. Beads were briefly washed 1×400 μLmore with TE-50 mM NaCl then 1×400 μL with MBG-Water.

To pre-hybridize the template DNA to the washed Primer-ConjugatedAgarose Beads, the template DNA was first prepared by serial dilution asfollows: The template DNA solutions were 1:1 mixtures of theaforementioned human p53 and human GST A2 fragments (i.e. 50% GST A2 and50% p53). To prepare these solutions, the Concentrated Stock TemplateDNA Solutions for human p53 and human GST A2, prepared as describedearlier in this Example, were subsequently diluted to 1 ng/μL inMBG-Water. The resultant 1 ng/μL human p53 and human GST A2 solutionswere the mixed together at a 1:1 ratio. This template mixture wasfurther diluted to 0.1 ng/μL in MBG-Water.

Next, the entire washed pellet of Primer-Conjugated Agarose Beads wasthen resuspended in 169.1 μL of a commercially available pre-mixed PCRreaction solution containing everything needed for PCR except templateDNA and primers (Platinum® PCR SuperMix High Fidelity; contains 22 U/mLcomplexed recombinant Taq DNA polymerase, Pyrococcus species GB-Dthermostable polymerase, Platinum® Taq Antibody, 66 mM Tris-SO₄ pH 8.9,19.8 mM (NH₄)₂SO₄, 2.4 mM MgSO₄, 220 μM dNTPs and stabilizers;Invitrogen Corporation, Carlsbad, Calif.; solution used at 92% strengthwith remaining 8% volume being MBG-Water). 53.3 μL portions of theresultant bead suspension (containing 4.3 μL actual bead volume), whichcontained no soluble primers and no template DNA, was divided intoseparate 0.5 mL polypropylene thin-wall PCR tubes which alreadycontained 1 μL of either 0 ng/μL (MBG-Water), 0.1 ng/mL or 1 ng/μL ofthe aforementioned template mixtures. This resulted in a ratio of 0, 180and 1,800 attomoles of template per μL of actual Primer-ConjugatedAgarose Bead volume. With 1 μL of Primer-Conjugated Agarose Beadsdetermined to contain approximately 1,000 beads, 180 and 1,800 attomolesof template per μL of beads represents a ratio of approximately 100,000and 1,000,000 template molecules added per each bead respectively (beadsphysically enumerated under a microscope both in diluted droplets ofbead suspension and with suspensions in a hemacytometer cell countingchamber). The resultant bead suspensions, now containing added templatebut no soluble (free) primers (only bead-bound primers), were thentreated as follows in a PCR machine (Mastercycler Personal; EppendorfAG, Hamburg, Germany) (lid temperature 105° C. and no mineral oil used):5 min 95° C. (denaturing) (beads were resuspended by brief gentle vortexmixing just before and at 2.5 min of this step), ramp down to 59° C. ata rate of 0.1° C./sec then hold 1 hour at 59° C. (annealing/capture oftemplate onto beads) (beads were resuspended by brief gentle vortexmixing at time zero of the 1 hour step and every 10 min thereafter), 10min 68° C. (fully extend any hybridized template-primer complexes once;no mixing). Immediately upon completion of the previous steps above,while the tubes were still at 68° C., the tubes were immediatelytransferred from the PCR machine to a crushed ice water bath. 400 μL ofice cold MBG-Water was added to each tube and the suspensionstransferred to fresh Filtration Devices. Filtration was immediatelyperformed as described earlier in this Example and the filtratediscarded. Using the same Filtration Devices, the beads were brieflywashed 2×400 μL with room temperature MBG-Water. Beads were furtherwashed 2×400 μL for 2.5 min each with room temperature 0.1M NaOH, withconstant vigorous vortex mixing, in order to strip off any hybridizedbut non-covalently bound template DNA, leaving only covalently attachedunused and extended primers on the beads. The beads were then brieflywashed 3×400 μL with 10×TE (100 mM Tris, pH 8.0, 10 mM EDTA), in orderto neutralize the pH, followed by 3×400 μL with MBG-Water, in order toremove the components of the 10×TE which would interfere with subsequentPCR.

Following the final filtration step on the bead samples, each washedbead pellet was resuspended in 50 μL of the commercial pre-mixed PCRsolution (Platinum® PCR SuperMix High Fidelity; Invitrogen Corporation,Carlsbad, Calif.) which was again used at 92% strength as describedearlier in this Example and contains all necessary components for PCRexcept template DNA and primers. However, a fluorescence BODIPY-FL-dUTPreagent was also added to a 20 μM final concentration from themanufacturer's 1 mM stock (ChromaTide® BODIPY® FL-14-dUTP; InvitrogenCorporation, Carlsbad, Calif.), in order to achieve subsequentfluorescence labeling of the PCR amplicon (PCR product). The suspensionswere then recovered from their Filtration Devices into fresh 0.5 mLpolypropylene thin-wall PCR tubes and subjected to the followingthermocycling in a PCR machine (Mastercycler Personal; Eppendorf AG,Hamburg, Germany) (lid temperature 105° C. and no mineral oil used): Aninitial denaturing step of 94° C. for 2 min (once) (beads were brieflyresuspended by gentle vortex mixing just before and at the end of thisstep), and 40 cycles of 94° C. for 30 sec (denature), 59° C. for 30 sec(anneal) and 68° C. for 2 min (extend); followed by a final extensionstep of 68° C. for 10 min (once).

400 μL of TE-50 mM NaCl-T (10 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl,0.01% v/v Tween-20) was added to each completed solid-phase bridge PCRreaction and the suspensions transferred to fresh 0.5 mL polypropylenePCR tubes. The beads were then spun down in a micro-centrifuge (justuntil reaches maximum speed of ˜13,000 rpm corresponding to ˜16,000×g)and the fluid supernatant carefully removed. The beads were washed 3×400μL more with TE-50 mM NaCl-T; resuspending by ˜5 sec vortex mixing thenspinning down and discarding the fluid supernatant as above. Followingthe final wash, as much of the fluid supernatant as possible was removedfrom the bead pellet by manual pipetting, with the beads going nearly todryness. The beads were lastly resuspended to 5% (v/v) using SP-PCRStorage Buffer (10 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl, all in 50%v/v glycerol). These intermediate beads could be stored at −20° C. andportions were subsequently used for a full second round of PCRthermocycling as described below.

Second Round of Solid-Phase Bridge PCR:

A portion of the above beads, following completion of the aforementionedinitial full round of solid-phase bridge PCR thermocycling (i.e. allpreceding steps), were subjected to a second full round of PCRthermocycling. To do so, 20 μL of the aforementioned 5% bead suspension(1 μL actual bead volume) was washed 2×400 μL with MBG-Water using aFiltration Device. Following the final filtration step on the beadsamples, each washed bead pellet was resuspended in 50 μL of thecommercial pre-mixed PCR solution (Platinum® PCR SuperMix High Fidelity;Invitrogen Corporation, Carlsbad, Calif.) which was again used at 92%strength as described earlier in this Example and contains all necessarycomponents for PCR except template DNA and primers. The fluorescenceBODIPY-FL-dUTP reagent was also added to a 20 μM final concentrationfrom the manufacturer's 1 mM stock (ChromaTide® BODIPY® FL-14-dUTP;Invitrogen Corporation, Carlsbad, Calif.), in order to achievesubsequent fluorescence labeling of the PCR amplicon (PCR product). Thesuspensions were then recovered from their Filtration Devices into fresh0.5 mL polypropylene thin-wall PCR tubes and subjected to the followingthermocycling in a PCR machine (Mastercycler Personal; Eppendorf AG,Hamburg, Germany) (lid temperature 105° C. and no mineral oil used): Aninitial denaturing step of 94° C. for 2 min (beads were brieflyresuspended by gentle vortex mixing just before and at the end of thisstep), and 40 cycles of 94° C. for 30 sec (denature), 59° C. for 30 sec(anneal) and 68° C. for 2 min (extend), followed by a final extensionstep of 68° C. for 10 min.

400 μL of TE-50 mM NaCl-T (10 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl,0.01% v/v Tween-20) was added to each completed solid-phase bridge PCRreaction and the suspensions transferred to fresh 0.5 mL polypropylenePCR tubes. The beads were then spun down in a micro-centrifuge (justuntil reaches maximum speed of ˜13,000 rpm corresponding to ˜16,000×g)and the fluid supernatant carefully removed. The beads were washed 3×400μL more with TE-50 mM NaCl-T; resuspending by ˜5 sec vortex mixing thenspinning down and discarding the fluid supernatant as above. Followingthe final wash, as much of the fluid supernatant as possible was removedfrom the bead pellet by manual pipetting, with the beads going nearly todryness. The beads were lastly resuspended to 5% (v/v) using SP-PCRStorage Buffer (10 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl, all in 50%v/v glycerol). These final beads, referred to from here forward asPost-PCR Beads, could be stored at −20° C. and portions weresubsequently used for fluorescence analysis as detailed below in thisExample.

Embedding the Beads in a Polyacrylamide Film and Fluorescence Imaging:

Lastly, the beads were embedded in a polyacrylamide film on a microscopeslide and fluorescently imaged to detect the BODIPY-FL-dUTP labeling ofthe PCR amplicon (PCR product). First however, beads were stained with aNeutrAvidin-Cy5 fluorescence conjugate, which binds the bead-boundbiotin groups, to enable detection of all beads regardless of thepresence of PCR amplicon. To do so, the NeutrAvidin-Cy5 fluorescenceconjugate was prepared as described previously in Example 35. Followingcompletion of all prior solid-phase bridge PCR reaction steps in thisExample, 20 μL of the aforementioned 5% (v/v) suspension of Post-PCRBeads was taken (i.e. 1 μL Post-PCR Bead volume), combined with 100 μLof the NeutrAvidin-Cy5 conjugate (38 ng/mL in TE-50 mM NaCl-T) and mixedgently for 5 min. The beads were then spun down in a standardmicro-centrifuge (just until reaches maximum speed of ˜13,000 rpmcorresponding to ˜16,000×g) and the fluid supernatant removed. The beadswere washed 3×400 μL with TE-50 mM NaCl-T; resuspending by ˜5 sec vortexmixing then spinning down and discarding the fluid supernatant as above.

After removing the final wash, the beads were embedded in apolyacrylamide film on a microscope slide and fluorescently imaged. Todo so, an Acrylamide Mix was prepared by combining the followingreagents in order: 244 μL of TE-50 mM NaCl, 57 μL of 40% acrylamide(19:1 cross-linking) (Bio-Rad Laboratories, Hercules, Calif.), 0.5 μLTEMED (Bio-Rad Laboratories, Hercules, Calif.), and 1 μL of a 10% (w/v)ammonium persulfate stock (prepared in MBG-Water from powder obtainedfrom Bio-Rad Laboratories, Hercules, Calif.). Each washed bead pelletwas then resuspended in 50 μL of the above Acrylamide Mix and combinedby brief vortex mixing. 25 μL of the bead suspension was then pipettedto a standard glass microscope slide and overlaid with a standard 18 mmsquare microscope cover glass (coverslip). Polymerization was allowed tooccur for ˜10 min protected from light. Note that the adequately slowpolymerization process allows all beads to settle to the surface of themicroscope slide by unit gravity. When polymerization was complete,imaging was performed using an ArrayWoRx^(e) BioChip fluorescencemicroarray reader (Applied Precision, LLC, Issaquah, Wash.).

Results:

A representative field-of-view of the raw fluorescence image is shown inFIG. 33A for all 3 sample permutations, as green and red 2-colorfluorescence image overlays for each. The minus template samplepermutation (−Template) was prepared in the same manner as the othersample permutations except that only the template DNA was omitted fromthe solid-phase bridge PCR reaction. Qualitatively, it is observed thatin the minus template negative control, virtually no beads havedetectible amplicon as evidenced by the lack of green fluorescencesignal from the BODIPY-FL-dUTP labeling, while all beads are detected bytheir independent red NeutrAvidin-Cy5 label. Note that a very lowpercentage of beads in the minus template negative control do havesignificant BODIPY-FL-dUTP labeling, which is believed to benon-specific amplification of non-template contaminant DNA oramplification of offset primer-dimers (so-called “false positives”).Nonetheless, at 180 and 1,800 attomoles of template per each μL of beadvolume, amplicon is observed on a significantly larger percentage of thebeads in comparison to the minus template negative control and at anoverall greater intensity of the BODIPY-FL-dUTP (green) signal. However,significant heterogeneity in the BODIPY-FL-dUTP (green) signal strengthis observed from bead-to-bead in the samples that received template.Note that all beads in all sample permutations have similar (uniform)red signal intensity to that of the minus template negative control (seebelow), but at higher amplicon levels, the red is masked by the greensignal in the image presented. It is also important to note that thedata shown in FIG. 33A is after the second round of solid-phase bridgePCR, and that no significant detectible BODIPY-FL-dUTP (green) signalwas observed on the beads after the first round of solid-phase bridgePCR (see methods portion of this Example for details of the first andsecond rounds of solid-phase bridge PCR).

For more precise data interpretation, the non-overlaid fluorescencegrayscale images were quantified by computer-assisted image analysisusing the ImageQuant software package (Molecular Dynamics; AmershamBiosciences Corp., Piscataway, N.J.). Average fluorescence intensitiesfor each bead (henceforth referred to as “bead intensity”) weredetermined in both the green and red fluorescence channels (i.e. averagefluorescence intensity over the entire area of a given individual bead).More than 350 beads were quantified for each sample permutation and thedata graphed in bar chart form (each bar in the graph represents thebead intensity of a specific individual bead) (FIG. 33B). Note that thered bead intensities alone were highly consistent from bead-to-bead inall sample permutations, as expected (not shown in FIG. 33B); if the redbead intensities for all beads in the minus template negative controlare averaged and normalized to 100%, the minus template negative controlis 100±12% (n=353 beads), in comparison, the 180 attomoles/μL beadssample averaged 99±9% (n=517 beads) and the 1,800 attomoles/μL beadssample averaged 99±8% (n=512 beads) of the minus template negativecontrol. Note that the fluorescence detector was not saturated in anycase.

The green bead intensity, corresponding to the level of amplicon, wasnormalized to the red bead intensity (i.e. the green to red ratio wascalculated for each bead), since the red bead intensity (biotin labelinglevel) is assumed to be proportional to each bead's binding capacity.The green to red ratios for all beads in the minus template negativecontrol averaged 1±1. Based on the data patterns and background levels,the following bead scoring parameters were used: Beads were scored as“strong positive” if the green to red ratio was ≧10 (red line in barchart of FIG. 33B), thereby corresponding to a signal-to-noise ratio of≧10:1 since the green to red ratio for the minus template negativecontrol (noise) averaged 1. Using these criteria, 4% of the beads scoreas “strong positive” in the 180 attomoles/μL of beads sample and 38% inthe 1,800 attomoles/μL of beads sample, in strong agreement with the10-fold difference in added template. It is critical to note that the“strong positives” in both the 180 and 1,800 attomoles template per μLof beads samples had comparable green to red ratios per each bead,averaging at 15±6 and 13±4 respectively; thus the amplicon levels in all“strong positives” of either sample were similar. Under these samecriteria, the minus template negative control had 0% “strong positives”.

Conversely, beads were scored as “negative” if their green to red ratiowas less than or equal to the average green to red ratio for the minustemplate negative control plus one standard deviation of the minustemplate negative control (i.e. green to red ratio of ≦2 is “negative”).Under these criteria, 29% of the beads score as “negative” in the 180attomoles/μL of beads sample and 3% in the 1,800 attomoles/μL of beadssample, again in strong agreement with the 10-fold difference in addedtemplate. Under these same criteria, the minus template negative controlhad 96% “negatives”. Together, these data suggest the amplification ofonly one or a few of the original template molecules per bead (e.g. 1-3copies per bead). Note also that there are “intermediately positive”beads that fall in between the “negative” and “strong positive” cutoffs.One possible explanation is that the “negative” beads amplified zerotemplate molecules, the “intermediately positive” beads 1 templatemolecule and the “strong positive” 2 template molecules. Furtherevidence of amplification of only one or a few of the original templatemolecules per bead (e.g. 1-3 copies per bead) is provided in laterExamples, such as titrating the initially added template DNA below thelevel of 180 attomoles/μL of beads (Example 37) as well assimultaneously detecting the human p53 and human GST A2 amplicons ondifferent beads with gene-specific oligonucleotide hybridization probeshaving different fluorescent labels (Examples 38 and 39).

Example 37 Effective Single Template Molecule Solid-Phase Bridge PCR andAmplicon Detection Through Fluorescence dUTP Labeling During the PCRReaction: Lower Limits of the Added Template Amount

This Example is similar to Example 36, repeating the 180 attomoles oftemplate per μL of beads permutation and further including a permutationof 18 attomoles of template per μL of beads, to demonstrate the lowerlimits of template concentration in this particular model system.

Preparing the Solid-Phase Bridge PCR Template DNA:

Performed as in Example 36.

Preparation of Agarose Beads Covalently Conjugated to PCR Primers Usedfor Solid-Phase Bridge PCR:

Performed as in Example 36.

Qualitative Analysis of Primer Attachment:

Performed as in Example 36.

First Round of Effective Single Template Molecule Solid-Phase BridgePCR:

Performed essentially as in Example 36, with slight modifications. Thefull protocol was as follows: 10 μL actual bead volume of the previouslyprepared Primer-Conjugated Agarose Beads was used per each sample, butfirst, each of the 10 μL of beads was washed separately in parallel,with heating. To do so, 50 μL each of the aforementioned 20% (v/v)Primer-Conjugated Agarose Bead suspension (10 μL actual bead volume) wasplaced into a 0.5 mL polypropylene thin-wall PCR tube. The beads werespun down briefly in a standard micro-centrifuge (just until reachesmaximum speed of ˜13,000 rpm corresponding to ˜16,000×g). As much of thefluid supernatant was removed as possible by manual pipetting, with thebeads nearly going to dryness. 40 μL each of TE-50 mM NaCl (10 mM Tris,pH 8.0, 1 mM EDTA, 50 mM NaCl) was added to the pellet, to bring thevolume back to the original 20% beads (v/v). The beads were brieflyvortex mixed then spun down and all fluid removed as described before.40 μL each of TE-50 mM NaCl was again added to the pellet as above andthe tube placed in a PCR machine (Mastercycler Personal; Eppendorf AG,Hamburg, Germany) at 95° C. for 10 min (lid temperature 105° C. and nomineral oil used) (beads were resuspended by brief gentle vortex mixingjust before and at 5 min of this step). After heating, the tube wasimmediately removed from the PCR machine, the beads diluted in 400 μL ofTE-50 mM NaCl and the bead suspension then transferred to a FiltrationDevice (see Example 36). Filtration was performed and the filtratediscarded. Beads were briefly washed 1×400 μL more with TE-50 mM NaClthen 1×400 μL with MBG-Water. Each set of beads was then resuspended in50 μL MBG-Water and transferred to a 0.5 mL polypropylene thin-wall PCRtube. The beads were spun down briefly in a standard micro-centrifuge(just until reaches maximum speed of ˜13,000 rpm corresponding to˜16,000×g). As much of the fluid supernatant was removed as possible bymanual pipetting, with the beads nearly going to dryness.

Next, to pre-hybridize the template DNA to the washed Primer-ConjugatedAgarose Beads, each pellet was then resuspended in 5 μL of dilutedtemplate solution, which contained no soluble primers. The p53 and GSTA2 template mixture was prepared to 1 ng/μL as described in Example 36(except 75% GST A2 and 25% p53). This template mixture was furtherserially diluted to 0.05 and 0.005 ng/μL in a commercially availablepre-mixed PCR reaction solution containing everything needed for PCRexcept template DNA and primers (Platinum® PCR SuperMix High Fidelity;contains 22 U/mL complexed recombinant Taq DNA polymerase, Pyrococcusspecies GB-D thermostable polymerase, Platinum® Taq Antibody, 66 mMTris-SO₄ pH 8.9, 19.8 mM (NH₄)₂SO₄, 2.4 mM MgSO₄, 220 μM dNTPs andstabilizers; Invitrogen Corporation, Carlsbad, Calif.; solution usedwithout prior dilution). This resulted in a ratio of 180 and 18attomoles of template per μL of actual Primer-Conjugated Agarose Beadvolume. With 1 μL of Primer-Conjugated Agarose Beads determined tocontain approximately 1,000 beads, 180 and 18 attomoles of template perμL of beads represents a ratio of approximately 100,000 and 10,000template molecules added per bead (beads physically enumerated under amicroscope both in diluted droplets of bead suspension and withsuspensions in a hemacytometer cell counting chamber). A minus templatenegative control was also prepared. The bead suspensions were only mixedmanually by gentle stirring with a pipette tip.

The resultant bead suspensions, now containing added template but nosoluble (free) primers (only bead-bound primers), were then treated asfollows in a PCR machine (Mastercycler Personal; Eppendorf AG, Hamburg,Germany) (lid temperature 105° C. and no mineral oil used): 5 min 95° C.(denaturing), ramp down to 59° C. at a rate of 0.1° C./sec then hold 1hour at 59° C. (annealing/capture of template onto beads), 10 min 68° C.(fully extend any hybridized template-primer complexes once; no mixing).Immediately upon completion of the previous steps above, while the tubeswere still at 68° C., the tubes were immediately transferred from thePCR machine to a crushed ice water bath. 400 μL of ice cold MBG-Waterwas added to each tube, the suspensions transferred to fresh FiltrationDevices, filtration was immediately performed and the filtrate discarded(see Example 36). Using the same Filtration Devices, the beads werebriefly washed 2×400 μL with room temperature MBG-Water. Beads werefurther washed 2×400 μL for 2.5 min each with room temperature 0.1 MNaOH, with constant vigorous vortex mixing, in order to strip off anyhybridized but non-covalently bound template DNA, leaving onlycovalently attached unused and extended primers on the beads. The beadswere then briefly washed 3×400 μL with 10×TE (100 mM Tris, pH 8.0, 10 mMEDTA), in order to neutralize the pH, followed by 3×400 μL withMBG-Water, in order to remove the components of the 10×TE which wouldinterfere with subsequent PCR.

Following the final filtration step on the bead samples, each washedbead pellet was resuspended in 100 μL of the commercial pre-mixed PCRsolution (Platinum® PCR SuperMix High Fidelity; Invitrogen Corporation,Carlsbad, Calif.) which was used at 92% strength (diluted withMBG-Water) and contains all necessary components for PCR except templateDNA and primers. However, since it was determined in Example 36 that nodetectible BODIPY-FL-dUTP fluorescence signal was observed after thefirst round of effective single template molecule solid-phase bridgePCR, the BODIPY-FL-dUTP reagent omitted from the PCR reaction at thisstage. The suspensions were then recovered from their Filtration Devicesinto fresh 0.5 mL polypropylene thin-wall PCR tubes and subjected to thefollowing thermocycling in a PCR machine (Mastercycler Personal;Eppendorf AG, Hamburg, Germany) (lid temperature 105° C. and no mineraloil used): An initial denaturing step of 94° C. for 2 min (once) (beadswere briefly resuspended by gentle vortex mixing just before and at theend of this step), and 40 cycles of 94° C. for 30 sec (denature), 59° C.for 30 sec (anneal) and 68° C. for 2 min (extend); followed by a finalextension step of 68° C. for 10 min (once).

400 μL of TE-50 mM NaCl-T (10 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl,0.01% v/v Tween-20) was added to each completed solid-phase bridge PCRreaction and the suspensions transferred to fresh 0.5 mL polypropylenePCR tubes. The beads were then spun down in a micro-centrifuge (justuntil reaches maximum speed of ˜13,000 rpm corresponding to ˜16,000×g)and the fluid supernatant carefully removed. The beads were washed 3×400μL more with TE-50 mM NaCl-T; resuspending by ˜5 sec vortex mixing thenspinning down and discarding the fluid supernatant as above. Followingthe final wash, as much of the fluid supernatant as possible was removedfrom the bead pellet by manual pipetting, with the beads going nearly todryness. The beads were lastly resuspended to 5% (v/v) using SP-PCRStorage Buffer (10 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl, all in 50%v/v glycerol). These intermediate beads could be stored at −20° C. andportions were subsequently used for a full second round of PCRthermocycling as described below.

Second Round of Solid-Phase Bridge PCR:

Performed as described in Example 36 except that a 5 μL portion of beads(actual bead volume) was used in 100 μL of the commercially availablepre-mixed PCR reaction solution (with the BODIPY-FL-dUTP labelingreagent).

Embedding the Beads in a Polyacrylamide Film and Fluorescence Imaging:

Performed as in Example 36.

Results:

A representative field-of-view of the raw fluorescence image is shown inFIG. 34A for all 3 sample permutations, as green and red 2-colorfluorescence image overlays for each. The minus template samplepermutation (−Template) was prepared in the same manner as the othersample permutations except that only the template DNA was omitted fromthe solid-phase bridge PCR reaction. Qualitatively, it is observed thatin the minus template negative control, virtually no beads havedetectible amplicon as evidenced by the lack of green fluorescencesignal from the BODIPY-FL-dUTP labeling, while all beads are detected bytheir independent red NeutrAvidin-Cy5 label. Note that a very lowpercentage of beads in the minus template negative control do havesignificant BODIPY-FL-dUTP labeling, which is believed to benon-specific amplification of non-template contaminant DNA oramplification of offset primer-dimers (so-called “false positives”). At18 attomoles of template per each μL of bead volume, the beads areindistinguishable from those of the minus template negative control.However, as in the previous Example 36, at 180 attomoles of template pereach μL of bead volume, amplicon is observed on a significantly largerpercentage of the beads in comparison to the minus template negativecontrol and at an overall greater intensity of the BODIPY-FL-dUTP(green) signal. Significant heterogeneity in the BODIPY-FL-dUTP (green)signal strength is observed from bead-to-bead in this sample. Note thatall beads in all sample permutations have similar (uniform) red signalintensity to that of the minus template negative control (see below),but at higher amplicon levels, the red is masked by the green signal inthe image presented.

For more precise data interpretation, the non-overlaid fluorescencegrayscale images were quantified by computer-assisted image analysisusing the ImageQuant software package (Molecular Dynamics; AmershamBiosciences Corp., Piscataway, N.J.). Average fluorescence intensitiesfor each bead (henceforth referred to as “bead intensity”) weredetermined in both the green and red fluorescence channels (i.e. averagefluorescence intensity over the entire area of a given individual bead).More than 350 beads were quantified for each sample permutation and thedata graphed in bar chart form (1 bar=1 bead) (FIG. 34B). As determinedin Example 36, the red bead intensities alone were highly consistentfrom bead-to-bead in all sample permutations, as expected (not shown inFIG. 34B).

The green bead intensity, corresponding to the level of amplicon, wasnormalized to the red bead intensity (i.e. the green to red ratio wascalculated for each bead), since the red bead intensity (biotin labelinglevel) is assumed to be proportional to each bead's binding capacity.The beads were scored as “strong positive” or “negative” as describedpreviously in Example 36. Using those criteria, 5% of the beads score as“strong positive” and 55% score as “negative” in the 180 attomoles/μL ofbeads sample, comparable to that observed previously in Example 36. Notealso that there are “intermediately positive” beads that fall in betweenthe “negative” and “strong positive” cutoffs (see Example 36 fordetails). Conversely, the 18 attomoles/μL of beads sample had only 1%“strong positives” and 96% “negatives” and was indistinguishable fromthe minus template negative control which had 1% “strong positives”(so-called “false positives”) and 95% “negatives”. This indicates that180 attomoles/mL of beads of initially added template, corresponding toroughly 100,000 template molecules initially added per bead (making noassumptions about the efficiency of template capture), is the lowerlimit of template concentration for this particular system.

Example 38 Effective Single Template Molecule Solid-Phase Bridge PCR:Validation of Effective Amplification of Single Template Molecules perBead Using 2 Template Species

This Example is similar to Example 37, except that the putative targettemplate concentration of 180 attomoles of template per μL of beads isconfirmed using dual fluorescence oligonucleotide hybridization probingto detect the levels of each of the 2 distinct amplicon species on eachbead.

Preparing the Solid-Phase Bridge PCR Template DNA:

Performed as in Example 36.

Preparation of Agarose Beads Covalently Conjugated to PCR Primers Usedfor Solid-Phase Bridge PCR

Performed as in Example 36.

Qualitative Analysis of Primer Attachment:

Performed as in Example 36.

First Round of Effective Single Template Molecule Solid-Phase BridgePCR:

Performed essentially as in Example 36, with slight modifications. Thefull protocol was as follows: 10 μL actual bead volume of the previouslyprepared Primer-Conjugated Agarose Beads was used per each sample, butfirst, each of the 10 μL of beads was washed separately in parallel,with heating. To do so, 50 μL each of the aforementioned 20% (v/v)Primer-Conjugated Agarose Bead suspension (10 μL actual bead volume) wasplaced into a 0.5 mL polypropylene thin-wall PCR tube. The beads werespun down briefly in a standard micro-centrifuge (just until reachesmaximum speed of ˜13,000 rpm corresponding to ˜16,000×g). As much of thefluid supernatant was removed as possible by manual pipetting, with thebeads nearly going to dryness. 40 μL each of TE-50 mM NaCl (10 mM Tris,pH 8.0, 1 mM EDTA, 50 mM NaCl) was added to the pellet, to bring thevolume back to the original 20% beads (v/v). The beads were brieflyvortex mixed then spun down and all fluid removed as described before.40 μL each of TE-50 mM NaCl was again added to the pellet as above andthe tube placed in a PCR machine (Mastercycler Personal; Eppendorf AG,Hamburg, Germany) at 95° C. for 10 min (lid temperature 105° C. and nomineral oil used) (beads were resuspended by brief gentle vortex mixingjust before and at 5 min of this step). After heating, the tube wasimmediately removed from the PCR machine, the beads diluted in 400 μL ofTE-50 mM NaCl and the bead suspension then transferred to a FiltrationDevice (see Example 36). Filtration was performed and the filtratediscarded. Beads were briefly washed 1×400 μL more with TE-50 mM NaClthen 1×400 μL with MBG-Water. Each set of beads was then resuspended in50 μL MBG-Water and transferred to a 0.5 mL polypropylene thin-wall PCRtube. The beads were spun down briefly in a standard micro-centrifuge(just until reaches maximum speed of ˜13,000 rpm corresponding to˜16,000×g). As much of the fluid supernatant was removed as possible bymanual pipetting, with the beads nearly going to dryness.

Next, to pre-hybridize the template DNA to the washed Primer-ConjugatedAgarose Beads, each pellet was then resuspended in 5 μL of dilutedtemplate solution, which contained no soluble primers. The p53 and GSTA2 template mixture was prepared to 1 ng/μL as described in Example 36(except 75% GST A2 and 25% p53). This template mixture was furtherserially diluted to 0.05 ng/μL in a commercially available pre-mixed PCRreaction solution containing everything needed for PCR except templateDNA and primers (Platinum® PCR SuperMix High Fidelity; contains 22 U/mLcomplexed recombinant Taq DNA polymerase, Pyrococcus species GB-Dthermostable polymerase, Platinum® Taq Antibody, 66 mM Tris-SO₄ pH 8.9,19.8 mM (NH₄)₂SO₄, 2.4 mM MgSO₄, 220 μM dNTPs and stabilizers;Invitrogen Corporation, Carlsbad, Calif.; solution used without priordilution). This resulted in a ratio of 180 attomoles of template per μLof actual Primer-Conjugated Agarose Bead volume. With 1 μL ofPrimer-Conjugated Agarose Beads determined to contain approximately1,000 beads, 180 attomoles of template per μL of beads represents aratio of approximately 100,000 template molecules added per bead (beadsphysically enumerated under a microscope both in diluted droplets ofbead suspension and with suspensions in a hemacytometer cell countingchamber). A minus template negative control was also prepared. The beadsuspensions were only mixed manually by gentle stirring with a pipettetip.

The resultant bead suspensions, now containing added template but nosoluble (free) primers (only bead-bound primers), were then treated asfollows in a PCR machine (Mastercycler Personal; Eppendorf AG, Hamburg,Germany) (lid temperature 105° C. and no mineral oil used): 5 min 95° C.(denaturing), ramp down to 59° C. at a rate of 0.1° C./sec then hold 1hour at 59° C. (annealing/capture of template onto beads), 10 min 68° C.(fully extend any hybridized template-primer complexes once; no mixing).Immediately upon completion of the previous steps above, while the tubeswere still at 68° C., the tubes were immediately transferred from thePCR machine to a crushed ice water bath. 400 μL of ice cold MBG-Waterwas added to each tube, the suspensions transferred to fresh FiltrationDevices, filtration was immediately performed and the filtrate discarded(see Example 36). Using the same Filtration Devices, the beads werebriefly washed 2×400 μL with room temperature MBG-Water. Beads werefurther washed 2×400 μL for 2.5 min each with room temperature 0.1MNaOH, with constant vigorous vortex mixing, in order to strip off anyhybridized but non-covalently bound template DNA, leaving onlycovalently attached unused and extended primers on the beads. The beadswere then briefly washed 3×400 μL with 10×TE (100 mM Tris, pH 8.0, 10 mMEDTA), in order to neutralize the pH, followed by 3×400 μL withMBG-Water, in order to remove the components of the 10×TE which wouldinterfere with subsequent PCR.

Following the final filtration step on the bead samples, each washedbead pellet was resuspended in 100 μL of the commercial pre-mixed PCRsolution (Platinum® PCR SuperMix High Fidelity; Invitrogen Corporation,Carlsbad, Calif.) which was used at 92% strength (diluted withMBG-Water) and contains all necessary components for PCR except templateDNA and primers. The BODIPY-FL-dUTP labeling reagent was not used in thesolid-phase bridge PCR reaction. The suspensions were then recoveredfrom their Filtration Devices into fresh 0.5 mL polypropylene thin-wallPCR tubes and subjected to the following thermocycling in a PCR machine(Mastercycler Personal; Eppendorf AG, Hamburg, Germany) (lid temperature105° C. and no mineral oil used): An initial denaturing step of 94° C.for 2 min (once) (beads were briefly resuspended by gentle vortex mixingjust before and at the end of this step), and 40 cycles of 94° C. for 30sec (denature), 59° C. for 30 sec (anneal) and 68° C. for 2 min(extend); followed by a final extension step of 68° C. for 10 min(once).

400 μL of TE-50 mM NaCl-T (10 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl,0.01% v/v Tween-20) was added to each completed solid-phase bridge PCRreaction and the suspensions transferred to fresh 0.5 mL polypropylenePCR tubes. The beads were then spun down in a micro-centrifuge (justuntil reaches maximum speed of ˜13,000 rpm corresponding to ˜16,000×g)and the fluid supernatant carefully removed. The beads were washed 3×400μL more with TE-50 mM NaCl-T; resuspending by ˜5 sec vortex mixing thenspinning down and discarding the fluid supernatant as above. Followingthe final wash, as much of the fluid supernatant as possible was removedfrom the bead pellet by manual pipetting, with the beads going nearly todryness. The beads were lastly resuspended to 5% (v/v) using SP-PCRStorage Buffer (10 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl, all in 50%v/v glycerol). These intermediate beads could be stored at −20° C. andportions were subsequently used for a full second round of PCRthermocycling as described below.

Second Round of Solid-Phase Bridge PCR.

Performed as described in Example 36 and 37 except that a 2 μL portionof beads (actual bead volume) was used in 50 μL of the commerciallyavailable pre-mixed PCR reaction solution and without the BODIPY-FL-dUTPlabeling reagent (i.e. no BODIPY-FL-dUTP labeling at any stage).

Oligonucleotide Hybridization Probing:

Fluorescently labeled oligonucleotide probes were commercially customsynthesized and HPLC purified by the manufacturer (Sigma-Genosys, TheWoodlands, Tex.). The probes were reconstituted to 100 μM in MBG-Waterand further desalted using MicroSpin G-25 columns according to themanufacturer's instructions (Amersham Biosciences Corp., Piscataway,N.J.), except that the columns were pre-washed 2×350 μL with MBG-Waterprior to sample loading (to wash, columns were mixed briefly in theMBG-Water then spun 1 min in a standard micro-centrifuge at the properspeed). The probes were diluted to 5 μM final in TE-50 mM NaCl forhybridization experiments. Prior to use however, the 5 μM probe solutionwas pre-clarified by spinning 1 min at maximum speed on amicro-centrifuge (˜13,000 rpm or ˜16,000×g) and collecting the fluidsupernatant. The supernatant was then passed though a Filtration Device(see Example 36) and the filtrate saved for use as the probing solution.

In this Example, simultaneous dual probing was performed by creating asingle probing solution containing 5 μM of each probe, labeled on their5′ ends with the Cy3 or Cy5 fluorophores by the manufacturer(Sigma-Genosys, The Woodlands, Tex.). The gene-specific probes werecomplementary to an internal segment of the human p53 and GST A2amplicons and had the following sequences:

[SEQ NO. 29] Human p53: 5′[Cy5]CATTTTCAgACCTATggAAACTACTTC3′ [SEQ NO.30] Human GST A2: 5′[Cy3]AgAATggAgTCCATCCggTg3′

Following completion of all prior solid-phase bridge PCR reaction stepsin this Example, 20 μL of the aforementioned stored 5% (v/v) stock beadsuspension (i.e. 1 μL post-PCR stored beads) was taken and washed 2×400μL with TE-50 mM NaCl using a Filtration Device (see Example 36). In theFiltration Device, each 1 μL pellet corresponding to each sample wasresuspended in 25 μL of the aforementioned clarified 5 μM probesolution. The beads were resuspended by manual pipetting thentransferred to 0.5 mL polypropylene thin-wall PCR tubes. Hybridizationwas performed as follows in a PCR machine (Mastercycler Personal;Eppendorf AG, Hamburg, Germany) (lid temperature always 105° C., nomineral oil used): 5 min 95° C. (denature) (beads resuspended by vortexmixing just before and at 2.5 min) followed by ramping down to 55° C. ata rate of 0.1° C./sec and subsequently holding 1 hour at 55° C.(anneal).

Just at the end of the above 1 hour 55° C. (anneal) step, while thetubes were still at 55° C. and still in the PCR machine, each sample wasrapidly diluted with 400 μL of 55° C. TE-50 mM NaCl, the suspensionsimmediately transferred to a Filtration Device and filtrationimmediately performed. The filtrate was then discarded. The beads werewashed 3×400 μL more with room temperature TE-50 mM NaCl then 1×400 μLwith room temperature TE-100 mM NaCl (10 mM Tris, pH 8.0, 1 mM EDTA, 100mM NaCl). The beads were recovered from the Filtration Devices byresuspending the pellet in 50 μL of TE-100 mM NaCl and transferring to a0.5 mL polypropylene PCR tube. The beads were spun down in a standardmicro-centrifuge (just until reaches maximum speed of 13,000 rpmcorresponding to ˜16,000×g) and the fluid supernatant removed.

Embedding the Beads in a Polyacrylamide Film and Fluorescence Imaging:

Lastly, the beads were embedded in a polyacrylamide film on a microscopeslide and fluorescently imaged to detect the bound Cy3 and Cy5 labeledhybridization probes. To do so, an Acrylamide Mix was prepared bycombining the following reagents in order: 244 μL of TE-100 mM NaCl, 57μL of 40% acrylamide (19:1 cross-linking) (Bio-Rad Laboratories,Hercules, Calif.), 0.5 μL TEMED (Bio-Rad Laboratories, Hercules,Calif.), and 1 μL of a 10% (w/v) ammonium persulfate stock (prepared inMBG-Water from powder obtained from Bio-Rad Laboratories, Hercules,Calif.). Each aforementioned washed bead pellet was then resuspended in50 μL of the above Acrylamide Mix and combined by brief vortex mixing.25 μL of the bead suspension was then pipetted to a standard glassmicroscope slide and overlaid with a standard 18 mm square microscopecover glass (coverslip). Polymerization was allowed to occur for ˜10 minprotected from light. Note that the adequately slow polymerizationprocess allows all beads to settle to the surface of the microscopeslide by unit gravity. When polymerization was complete, imaging wasperformed using an ArrayWoRx^(e) BioChip fluorescence microarray reader(Applied Precision, LLC, Issaquah, Wash.).

Results:

A representative field-of-view of the fluorescence image is shown inFIG. 35 for both sample permutations, as green and red 2-colorfluorescence image overlays for each. The minus template samplepermutation (−Template) was prepared in the same manner as the othersample permutations except that only the template DNA was omitted fromthe solid-phase bridge PCR reaction. In the figure, green corresponds tothe GST A2 probe (Cy3) and red the p53 probe (Cy5). For the plustemplate sample permutation, non-overlaid green and red fluorescenceimages of the same selected region are also shown. Because the green andred signals arise from different binding probes (for GST A2 and p53)labeled with different fluorophores (Cy3 and Cy5), the two are notdirectly comparable with respect to relative quantification of the levelof GST A2 and p53 amplicon on each bead. This is due to potentiallydifferent probe binding efficiencies and differences in fluorescenceoutput and signal collection efficiencies. Therefore, for the imagepresented in FIG. 35, all image intensity levels of the red channel havebeen scaled linearly (uniformly) for normalization, such that themaximum intensity in the red channel matched the maximum intensity inthe green channel. Qualitatively, it is observed that in the minustemplate negative control, no beads have detectible amplicon asevidenced by the lack of any significant fluorescence signal. However,the presence of beads in the minus template negative control sample canbe confirmed by the extremely weak auto-fluorescence of the beadsthemselves, which have a uniform green:red fluorescence ratio when theimage is observed at very high contrast settings (beads appearinguniformly yellow-orange in the image overlay, shown in the inset box, inthe minus template negative control panel of FIG. 35). In the plustemplate sample, significant probing signal is observed for both GST A2(green) and p53 (red). The data suggest amplification of only 1 or a feworiginal template molecules per bead, otherwise, relatively uniformgreen:red (or visa versa) ratios would be expected from bead-to-bead.Instead, it is clear from the data that a sub-population of beads has asignificantly higher green:red signal ratio (elevated GST A2 content)compared to that of the other beads. Likewise, a differentsub-population of beads has a significantly higher red:green signalratio (elevated p53 content) compared to that of the other beads.Furthermore, the proportion of these 2 sub-populations approximates thatof the initially added template DNA mix (75% GST A2 and 25% p53). Thesubsequent Example 39 provides a more quantitative analysis of thisexperimental system.

Example 39 Effective Single Template Molecule Solid-Phase Bridge PCR:Validation of Effective Amplification of Single Template Molecules perBead by Titrating Ratios of 2 Template Species

This Example is similar to Example 38, except that the ratio of humanGST A2 and p53 in the initially added template mix was modulated.Following dual fluorescence oligonucleotide hybridization probing todetect the level of each amplicon on each bead, the beads werequantified and enumerated.

Preparing the Solid-Phase Bridge PCR Template DNA:

Performed as in Example 36.

Preparation of Agarose Beads Covalently Conjugated to PCR Primers Usedfor Solid-Phase Bridge PCR:

Performed as in Example 36.

Qualitative Analysis of Primer Attachment:

Performed as in Example 36.

First Round of Effective Single Template Molecule Solid-Phase BridgePCR:

Performed essentially as in Example 36, with slight modifications. Thefull protocol was as follows: 10 μL actual bead volume of the previouslyprepared Primer-Conjugated Agarose Beads was used per each sample, butfirst, each of the 10 μL of beads was washed separately in parallel,with heating. To do so, 50 μL each of the aforementioned 20% (v/v)Primer-Conjugated Agarose Bead suspension (10 μL actual bead volume) wasplaced into a 0.5 mL polypropylene thin-wall PCR tube. The beads werespun down briefly in a standard micro-centrifuge (just until reachesmaximum speed of ˜13,000 rpm corresponding to ˜16,000×g). As much of thefluid supernatant was removed as possible by manual pipetting, with thebeads nearly going to dryness. 40 μL each of TE-50 mM NaCl (10 mM Tris,pH 8.0, 1 mM EDTA, 50 mM NaCl) was added to the pellet, to bring thevolume back to the original 20% beads (v/v). The beads were brieflyvortex mixed then spun down and all fluid removed as described before.40 μL each of TE-50 mM NaCl was again added to the pellet as above andthe tube placed in a PCR machine (Mastercycler Personal; Eppendorf AG,Hamburg, Germany) at 95° C. for 10 min (lid temperature 105° C. and nomineral oil used) (beads were resuspended by brief gentle vortex mixingjust before and at 5 min of this step). After heating, the tube wasimmediately removed from the PCR machine, the beads diluted in 400 μL ofTE-50 mM NaCl and the bead suspension then transferred to a FiltrationDevice (see Example 36). Filtration was performed and the filtratediscarded. Beads were briefly washed 1×400 μL more with TE-50 mM NaClthen 1×400 μL with MBG-Water. Each set of beads was then resuspended in50 μL MBG-Water and transferred to a 0.5 mL polypropylene thin-wall PCRtube. The beads were spun down briefly in a standard micro-centrifuge(just until reaches maximum speed of 13,000 rpm corresponding to˜16,000×g). As much of the fluid supernatant was removed as possible bymanual pipetting, with the beads nearly going to dryness.

Next, to pre-hybridize the template DNA to the washed Primer-ConjugatedAgarose Beads, each pellet was then resuspended in 5 μL of dilutedtemplate solution, which contained no soluble primers. The p53 and GSTA2 template mixture was prepared to 1 ng/μL as described in Example 36(except at various ratios of p53 to GST A2). This template mixture wasfurther serially diluted to 0.05 ng/μL in a commercially availablepre-mixed PCR reaction solution containing everything needed for PCRexcept template DNA and primers (Platinum® PCR SuperMix High Fidelity;contains 22 U/mL complexed recombinant Taq DNA polymerase, Pyrococcusspecies GB-D thermostable polymerase, Platinum® Taq Antibody, 66 mMTris-SO₄ pH 8.9, 19.8 mM (NH₄)₂SO₄, 2.4 mM MgSO₄, 220 μM dNTPs andstabilizers; Invitrogen Corporation, Carlsbad, Calif.; solution usedwithout prior dilution). This resulted in a ratio of 180 attomoles oftemplate per μL of actual Primer-Conjugated Agarose Bead volume. With 1μL of Primer-Conjugated Agarose Beads determined to containapproximately 1,000 beads, 180 attomoles of template per μL of beadsrepresents a ratio of approximately 100,000 template molecules added perbead (beads physically enumerated under a microscope both in diluteddroplets of bead suspension and with suspensions in a hemacytometer cellcounting chamber). A minus template negative control was also prepared.The bead suspensions were only mixed manually by gentle stirring with apipette tip.

The resultant bead suspensions, now containing added template but nosoluble (free) primers (only bead-bound primers), were then treated asfollows in a PCR machine (Mastercycler Personal; Eppendorf AG, Hamburg,Germany) (lid temperature 105° C. and no mineral oil used): 5 min 95° C.(denaturing), ramp down to 59° C. at a rate of 0.1° C./sec then hold 1hour at 59° C. (annealing/capture of template onto beads), 10 min 68° C.(fully extend any hybridized template-primer complexes once; no mixing).Immediately upon completion of the previous steps above, while the tubeswere still at 68° C., the tubes were immediately transferred from thePCR machine to a crushed ice water bath. 400 μL of ice cold MBG-Waterwas added to each tube, the suspensions transferred to fresh FiltrationDevices, filtration was immediately performed and the filtrate discarded(see Example 36). Using the same Filtration Devices, the beads werebriefly washed 2×400 μL with room temperature MBG-Water. Beads werefurther washed 2×400 μL for 2.5 min each with room temperature 0.1MNaOH, with constant vigorous vortex mixing, in order to strip off anyhybridized but non-covalently bound template DNA, leaving onlycovalently attached unused and extended primers on the beads. The beadswere then briefly washed 3×400 μL with 10×TE (100 mM Tris, pH 8.0, 10 mMEDTA), in order to neutralize the pH, followed by 3×400 μL withMBG-Water, in order to remove the components of the 10×TE which wouldinterfere with subsequent PCR.

Following the final filtration step on the bead samples, each washedbead pellet was resuspended in 100 μL of the commercial pre-mixed PCRsolution (Platinum® PCR SuperMix High Fidelity; Invitrogen Corporation,Carlsbad, Calif.) which was used at 92% strength (diluted withMBG-Water) and contains all necessary components for PCR except templateDNA and primers. The PCR reaction was further supplemented with 0.15U/μL final of additional PlatinumTaq DNA Polymerase High Fidelity addedfrom a 5 U/μL manufacturer's stock (Invitrogen Corporation, Carlsbad,Calif.). The BODIPY-FL-dUTP labeling reagent was not used in thesolid-phase bridge PCR reaction. The suspensions were then recoveredfrom their Filtration Devices into fresh 0.5 mL polypropylene thin-wallPCR tubes and subjected to the following thermocycling in a PCR machine(Mastercycler Personal; Eppendorf AG, Hamburg, Germany) (lid temperature105° C. and no mineral oil used): An initial denaturing step of 94° C.for 2 min (once) (beads were briefly resuspended by gentle vortex mixingjust before and at the end of this step), and 40 cycles of 94° C. for 30sec (denature), 59° C. for 30 sec (anneal) and 68° C. for 2 min(extend); followed by a final extension step of 68° C. for 10 min(once).

400 μL of TE-50 mM NaCl-T (10 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl,0.01% v/v Tween-20) was added to each completed solid-phase bridge PCRreaction and the suspensions transferred to fresh 0.5 mL polypropylenePCR tubes. The beads were then spun down in a micro-centrifuge (justuntil reaches maximum speed of ˜13,000 rpm corresponding to ˜16,000×g)and the fluid supernatant carefully removed. The beads were washed 3×400μL more with TE-50 mM NaCl-T; resuspending by ˜5 sec vortex mixing thenspinning down and discarding the fluid supernatant as above. Followingthe final wash, as much of the fluid supernatant as possible was removedfrom the bead pellet by manual pipetting, with the beads going nearly todryness. The beads were lastly resuspended to 5% (v/v) using SP-PCRStorage Buffer (10 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl, all in 50%v/v glycerol). These intermediate beads could be stored at −20° C. andportions were subsequently used for a full second round of PCRthermocycling as described below.

Second Round of Solid-Phase Bridge PCR:

Performed as described in Example 36 and 37 except that a 10 μL portionof beads (actual bead volume) was used in 100 μL of the commerciallyavailable pre-mixed PCR reaction solution and without the BODIPY-FL-dUTPlabeling reagent (i.e. no BODIPY-FL-dUTP labeling at any stage).Furthermore, the solid-phase bridge PCR reaction was furthersupplemented with 0.15 U/μL final of additional PlatinumTaq DNAPolymerase High Fidelity added from a 5 U/μL manufacturer's stock(Invitrogen Corporation, Carlsbad, Calif.).

Oligonucleotide Hybridization Probing:

Fluorescently labeled oligonucleotide probes were commercially customsynthesized and HPLC purified by the manufacturer (Sigma-Genosys, TheWoodlands, Tex.). The probes were reconstituted to 100 μM in MBG-Waterand further desalted using MicroSpin G-25 columns according to themanufacturer's instructions (Amersham Biosciences Corp., Piscataway,N.J.), except that the columns were pre-washed 2×350 μL with MBG-Waterprior to sample loading (to wash, columns were mixed briefly in theMBG-Water then spun 1 min in a standard micro-centrifuge at the properspeed). The probes were diluted to 5 μM final in TE-50 mM NaCl forhybridization experiments. Prior to use however, the 5 μM probe solutionwas pre-clarified by spinning 1 min at maximum speed on amicro-centrifuge (13,000 rpm or ˜16,000×g) and collecting the fluidsupernatant. The supernatant was then passed though a Filtration Device(see Example 36) and the filtrate saved for use as the probing solution.

In this Example, simultaneous dual probing was performed by creating asingle probing solution containing 5 μM of each probe, labeled on their5′ ends with the Cy3 or Cy5 fluorophores by the manufacturer(Sigma-Genosys, The Woodlands, Tex.). The gene-specific probes werecomplementary to an internal segment of the human p53 and GST A2amplicons and had the following sequences:

[SEQ NO. 31] Human p53: 5′[Cy5]CATTTTCAgACCTATggAAACTACTTC3′ [SEQ NO.32] Human GST A2: 5′[Cy3]AgAATggAgTCCATCCggTg3′

Following completion of all prior solid-phase bridge PCR reaction stepsin this Example, 20 μL of the aforementioned stored 5% (v/v) stock beadsuspension (i.e. 1 μL post-PCR stored beads) was taken and washed 2×400μL with TE-50 mM NaCl using a Filtration Device (see Example 36). In theFiltration Device, each 1 μL pellet corresponding to each sample wasresuspended in 25 μL of the aforementioned clarified 5 μM probesolution. The beads were resuspended by manual pipetting thentransferred to 0.5 mL polypropylene thin-wall PCR tubes. Hybridizationwas performed as follows in a PCR machine (Mastercycler Personal;Eppendorf AG, Hamburg, Germany) (lid temperature always 105° C., nomineral oil used): 5 min 95° C. (denature) (beads resuspended by vortexmixing just before and at 2.5 min) followed by ramping down to 55° C. ata rate of 0.1° C./sec and subsequently holding 1 hour at 55° C.(anneal).

Just at the end of the above 1 hour 55° C. (anneal) step, while thetubes were still at 55° C. and still in the PCR machine, each sample wasrapidly diluted with 400 μL of 55° C. TE-50 mM NaCl, the suspensionsimmediately transferred to a Filtration Device and filtrationimmediately performed. The filtrate was then discarded. The beads werewashed 1×400 μL more with room temperature TE-50 mM NaCl. Next, tofluorescently stain all beads independently of the presence or absenceof amplicon, the beads were treated 1× for 5 min with gentle mixingusing 200 μL of TE-50 mM NaCl containing 0.01% (v/v) Tween-20 and 50pg/μL of a streptavidin Alexa Fluor 488 conjugate (InvitrogenCorporation, Carlsbad, Calif.). The beads were then further washed 3×400μL with TE-100 mM NaCl (10 mM Tris, pH 8.0, 1 mM EDTA, 100 mM NaCl). Thebeads were recovered from the Filtration Devices by resuspending thepellet in 50 μL of TE-100 mM NaCl and transferring to a 0.5 mLpolypropylene PCR tube. The beads were spun down in a standardmicro-centrifuge (just until reaches maximum speed of ˜13,000 rpmcorresponding to ˜16,000×g) and the fluid supernatant removed.

Embedding the Beads in a Polyacrylamide Film and Fluorescence Imaging:

Lastly, the beads were embedded in a polyacrylamide film on a microscopeslide and fluorescently imaged to detect the bound Cy3 and Cy5 labeledoligonucleotide hybridization probes as well as the Alexa Fluor 488labeled total bead probe (detects all beads independent of eitheramplicon). To do so, an Acrylamide Mix was prepared by combining thefollowing reagents in order: 244 μL of TE-100 mM NaCl, 57 μL of 40%acrylamide (19:1 cross-linking) (Bio-Rad Laboratories, Hercules,Calif.), 0.5 μL TEMED (Bio-Rad Laboratories, Hercules, Calif.), and 1 μLof a 10% (w/v) ammonium persulfate stock (prepared in MBG-Water frompowder obtained from Bio-Rad Laboratories, Hercules, Calif.). Eachaforementioned washed bead pellet was then resuspended in 50 μL of theabove Acrylamide Mix and combined by brief vortex mixing. 25 μL of thebead suspension was then pipetted to a standard glass microscope slideand overlaid with a standard 18 mm square microscope cover glass(coverslip). Polymerization was allowed to occur for 10 min protectedfrom light. Note that the adequately slow polymerization process allowsall beads to settle to the surface of the microscope slide by unitgravity. When polymerization was complete, imaging was performed usingan ArrayWoRx^(e) BioChip fluorescence microarray reader (AppliedPrecision, LLC, Issaquah, Wash.).

Results:

A representative field-of-view of the fluorescence image is shown inFIG. 36A for all sample permutations, as blue, red and green 3-colorfluorescence image overlays for each. The minus template samplepermutation (−Template) was prepared in the same manner as the othersample permutations except that only the template DNA was omitted fromthe solid-phase bridge PCR reaction. In the figure, red corresponds tothe p53 probe (Cy5) and green the GST A2 probe (Cy3), while bluecorresponds to the Alexa Fluor 488 labeled total bead probe (detects allbeads independent of either amplicon). Because the red and green signalsarise from different binding probes (for p53 and GST A2) labeled withdifferent fluorophores (Cy5 and Cy3), the two are not directlycomparable with respect to relative quantification of the level of p53and GST A2 amplicon on each bead. This is due to potentially differentprobe binding efficiencies and differences in fluorescence output andsignal collection efficiencies. Therefore, for the image presented inFIG. 36A, all image intensity levels of the red channel have been scaledlinearly (uniformly) for normalization, such that the maximum intensityin the red channel matched the maximum intensity in the green channel.

The raw, unmodified, non-overlaid fluorescence grayscale images werequantified by computer-assisted image analysis using the ImageQuantsoftware package (Molecular Dynamics; Amersham Biosciences Corp.,Piscataway, N.J.). Average fluorescence intensities for each bead(henceforth referred to as “bead intensity”) were determined in both thered and green fluorescence channels (i.e. average fluorescence intensityover the entire area of a given individual bead). More than 700 beadswere quantified for each sample permutation. The beads were scored asfollows: The average bead intensity for all beads in the minus templatenegative control (i.e. the blank), for either the red or greenfluorescence channels, was taken as the “noise” level (i.e. background)for that given channel. The signal to noise ratio for all beads in theplus template sample permutations was calculated for both the red (p53)and green (GST A2) fluorescence channels. Beads were scored positive forp53 if the red signal to noise ratio was ÷10:1. Likewise, beads werescored positive for GST A2 if the green signal to noise ratio was ≧10:1.The number of p53 positive scores and GST A2 positive scores wasexpressed as a percent of the total positive scores. As showngraphically in FIG. 36B, when 50:50, 75:25 and 95:5 p53:GST A2 templatemixtures were used, actual ratios obtained of p53 positive scores to GSTA2 positive scores were 34:66, 76:24, and 97:3 respectively, in closecorrelation with the added template. While the 50:50 p53:GST A2 sampledeviated 16 percentage points from the expected (experimentalvariability), the 75:25 and 95:5 p53:GST A2 samples differed by no morethan 2 percentage points, for an average deviation of 6 percentagepoints.

These data suggest that only 1 or a few original template molecules havebeen amplified per bead, otherwise, relatively constant p53:GST A2 (orvisa versa) signal ratios from bead-to-bead within each samplepermutation would be expected. Furthermore, if amplification ofsignificantly more than 1 or a few original template molecules per beadwas occurring, decreasing GST A2 signal to noise ratios correlating withdecreasing amounts of GST A2 template across the various samplepermutations would be expected. Instead, the signal to noise ratios forpositively scoring GST A2 beads remains relatively constant, averaging42:1 and 56:1 for the 50:50 and 95:5 p53:GST A2 samples respectively;despite the 10-fold decrease in overall GST A2 template amount andnearly 20-fold decrease in relative GST A2 template abundance in the95:5 p53:GST A2 sample. While the signal to noise ratios remainconstant, the number of positively scoring GST A2 beads decreases in amanner consistent with the ratio of added template.

Example 40 Effective Single Template Molecule Solid-Phase Bridge PCR:Multiplexed Cell-Free Expression with In Situ Protein Capture, ContactPhoto-Transfer and Antibody Detection Preparing the Solid-Phase BridgePCR Template DNA:

Performed as in Example 36.

Preparation of Agarose Beads Covalently Conjugated to PCR Primers Usedfor Solid-Phase Bridge PCR:

Performed as in Example 36.

Qualitative Analysis of Primer Attachment:

Performed as in Example 36.

First Round of Effective Single Template Molecule Solid-Phase BridgePCR:

Performed essentially as in Example 36, with slight modifications. Thefull protocol was as follows: 10 μL actual bead volume of the previouslyprepared Primer-Conjugated Agarose Beads was used per each sample, butfirst, each of the 10 μL of beads was washed separately in parallel,with heating. To do so, 50 μL each of the aforementioned 20% (v/v)Primer-Conjugated Agarose Bead suspension (10 μL actual bead volume) wasplaced into a 0.5 mL polypropylene thin-wall PCR tube. The beads werespun down briefly in a standard micro-centrifuge (just until reachesmaximum speed of ˜13,000 rpm corresponding to ˜16,000×g). As much of thefluid supernatant was removed as possible by manual pipetting, with thebeads nearly going to dryness. 40 μL each of TE-50 mM NaCl (10 mM Tris,pH 8.0, 1 mM EDTA, 50 mM NaCl) was added to the pellet, to bring thevolume back to the original 20% beads (v/v). The beads were brieflyvortex mixed then spun down and all fluid removed as described before.40 μL each of TE-50 mM NaCl was again added to the pellet as above andthe tube placed in a PCR machine (Mastercycler Personal; Eppendorf AG,Hamburg, Germany) at 95° C. for 10 min (lid temperature 105° C. and nomineral oil used) (beads were resuspended by brief gentle vortex mixingjust before and at 5 min of this step). After heating, the tube wasimmediately removed from the PCR machine, the beads diluted in 400 μL ofTE-50 mM NaCl and the bead suspension then transferred to a FiltrationDevice (see Example 36). Filtration was performed and the filtratediscarded. Beads were briefly washed 1×400 μL more with TE-50 mM NaClthen 1×400 μL with MBG-Water. Each set of beads was then resuspended in50 μL MBG-Water and transferred to a 0.5 mL polypropylene thin-wall PCRtube. The beads were spun down briefly in a standard micro-centrifuge(just until reaches maximum speed of ˜13,000 rpm corresponding to˜16,000×g). As much of the fluid supernatant was removed as possible bymanual pipetting, with the beads nearly going to dryness.

Next, to pre-hybridize the template DNA to the washed Primer-ConjugatedAgarose Beads, each pellet was then resuspended in 5 μL of dilutedtemplate solution, which contained no soluble primers. The p53 and GSTA2 template mixture was prepared to 1 ng/μL as described in Example 36(except 75% GST A2 and 25% p53). This template mixture was furtherserially diluted to 0.05 ng/μL in a commercially available pre-mixed PCRreaction solution containing everything needed for PCR except templateDNA and primers (Platinum® PCR SuperMix High Fidelity; contains 22 U/mLcomplexed recombinant Taq DNA polymerase, Pyrococcus species GB-Dthermostable polymerase, Platinum® Taq Antibody, 66 mM Tris-SO₄ pH 8.9,19.8 mM (NH₄)₂SO₄, 2.4 mM MgSO₄, 220 μM dNTPs and stabilizers;Invitrogen Corporation, Carlsbad, Calif.; solution used without priordilution). This resulted in a ratio of 180 attomoles of template per μLof actual Primer-Conjugated Agarose Bead volume. With 1 μL ofPrimer-Conjugated Agarose Beads determined to contain approximately1,000 beads, 180 attomoles of template per μL of beads represents aratio of approximately 100,000 template molecules added per bead (beadsphysically enumerated under a microscope both in diluted droplets ofbead suspension and with suspensions in a hemacytometer cell countingchamber). A minus template negative control was also prepared. The beadsuspensions were only mixed manually by gentle stirring with a pipettetip.

The resultant bead suspensions, now containing added template but nosoluble (free) primers (only bead-bound primers), were then treated asfollows in a PCR machine (Mastercycler Personal; Eppendorf AG, Hamburg,Germany) (lid temperature 105° C. and no mineral oil used): 5 min 95° C.(denaturing), ramp down to 59° C. at a rate of 0.1° C./sec then hold 1hour at 59° C. (annealing/capture of template onto beads), 10 min 68° C.(fully extend any hybridized template-primer complexes once; no mixing).Immediately upon completion of the previous steps above, while the tubeswere still at 68° C., the tubes were immediately transferred from thePCR machine to a crushed ice water bath. 400 μL of ice cold MBG-Waterwas added to each tube, the suspensions transferred to fresh FiltrationDevices, filtration was immediately performed and the filtrate discarded(see Example 36). Using the same Filtration Devices, the beads werebriefly washed 2×400 μL with room temperature MBG-Water. Beads werefurther washed 2×400 μL for 2.5 min each with room temperature 0.1MNaOH, with constant vigorous vortex mixing, in order to strip off anyhybridized but non-covalently bound template DNA, leaving onlycovalently attached unused and extended primers on the beads. The beadswere then briefly washed 3×400 μL with 10×TE (100 mM Tris, pH 8.0, 10 mMEDTA), in order to neutralize the pH, followed by 3×400 μL withMBG-Water, in order to remove the components of the 10×TE which wouldinterfere with subsequent PCR.

Following the final filtration step on the bead samples, each washedbead pellet was resuspended in 100 μL of the commercial pre-mixed PCRsolution (Platinum® PCR SuperMix High Fidelity; Invitrogen Corporation,Carlsbad, Calif.) which was used at 92% strength (diluted withMBG-Water) and contains all necessary components for PCR except templateDNA and primers. The BODIPY-FL-dUTP labeling reagent was not used duringthe solid-phase bridge PCR. The suspensions were then recovered fromtheir Filtration Devices into fresh 0.5 mL polypropylene thin-wall PCRtubes and subjected to the following thermocycling in a PCR machine(Mastercycler Personal; Eppendorf AG, Hamburg, Germany) (lid temperature105° C. and no mineral oil used): An initial denaturing step of 94° C.for 2 min (once) (beads were briefly resuspended by gentle vortex mixingjust before and at the end of this step), and 40 cycles of 94° C. for 30sec (denature), 59° C. for 30 sec (anneal) and 68° C. for 2 min(extend); followed by a final extension step of 68° C. for 10 min(once).

400 μL of TE-50 mM NaCl-T (10 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl,0.01% v/v Tween-20) was added to each completed solid-phase bridge PCRreaction and the suspensions transferred to fresh 0.5 mL polypropylenePCR tubes. The beads were then spun down in a micro-centrifuge (justuntil reaches maximum speed of ˜13,000 rpm corresponding to ˜16,000×g)and the fluid supernatant carefully removed. The beads were washed 3×400μL more with TE-50 mM NaCl-T; resuspending by 5 sec vortex mixing thenspinning down and discarding the fluid supernatant as above. Followingthe final wash, as much of the fluid supernatant as possible was removedfrom the bead pellet by manual pipetting, with the beads going nearly todryness. The beads were lastly resuspended to 5% (v/v) using SP-PCRStorage Buffer (10 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl, all in 50%v/v glycerol). These intermediate beads could be stored at −20° C. andportions were subsequently used for a full second round of PCRthermocycling as described below.

Second Round of Solid-Phase Bridge PCR:

Performed as described in Example 36 and 37 except that a 2 μL portionof beads (actual bead volume) was used in 50 μL of the commerciallyavailable pre-mixed PCR reaction solution and without the BODIPY-FL-dUTPlabeling reagent (i.e. no BODIPY-FL-dUTP labeling at any stage).

Attaching the PC-Antibody to Beads Following Solid-phase bridge PCR:

Following the solid-phase bridge PCR reaction, 0.5 μL actual bead volumeper sample was washed briefly 3×400 μL with TE-Saline (10 mM Tris-HCl,pH 8.0, 1 mM EDTA, 200 mM NaCl). Unless otherwise noted, all washes andbead manipulations were performed in batch mode using 0.45 micron poresize, PVDF membrane, micro-centrifuge Filtration Devices to facilitatemanipulation of the beaded matrix (˜100 micron beads) and exchange thebuffers (Ultrafree-MC Durapore Micro-centrifuge Filtration Devices, 400μL capacity; Millipore, Billerica, Mass.). NeutrAvidin (tetrameric) wasthen attached to the bead bound biotin-amine linker, in excess, bytreatment with 200 μL of a 0.5 μg/μL solution in TE-Saline for 20 min(note: biotin-amine linker attached during previous preparation ofPrimer-Conjugated Agarose Beads; see Example 36). Beads were washedbriefly 4×400 μL with TE-Saline.

The beads were next coated with a monoclonal mouse anti-FLAG tag captureantibody which was converted to photocleavable form by conjugation toPC-biotin. Creation of the photocleavable antibody (PC-antibody) wasperformed similar to as described in Example 2. To first create thePC-antibody (prepared in advance), 1 mg of antibody as supplied by themanufacturer (Mouse Anti-FLAG M2 Antibody; Sigma-Aldrich, St. Louis,Mo.) was purified on a NAP-5 desalting column according to themanufacturer's instructions (Amersham Biosciences Corp., Piscataway,N.J.) against a 200 mM sodium bicarbonate and 200 mM NaCl buffer(nuclease-free reagents). The resultant antibody was then reacted with25 molar equivalents of AmberGen's PC-biotin-NHS labeling reagent (addedfrom a 50 mM stock in anhydrous DMF) for 30-60 min with gentle mixing.The labeled antibody was then purified on a NAP-10 desalting columnaccording to the manufacturer's instructions (Amersham BiosciencesCorp., Piscataway, N.J.) against TE-Saline buffer. This preparedmonoclonal anti-FLAG PC-biotin conjugate was then loaded onto the beadsby treatment of the beads with 250 μL of 0.15 μg/μL in TE-Saline for 20min. Beads were washed briefly 4×400 μL in TE-Saline followed by 2×400μL in Molecular Biology Grade Water (MBG-Water).

Multiplexed Cell-Free Expression of the Beads and In Situ ProteinCapture:

The 0.5 μL bead pellets were then resuspended in 25 μL of the E. colibased PureSystem cell-free expression mixture (mixture preparedaccording to the manufacturer's instructions; Post Genome Institute Co.,LTD., Japan) (no soluble DNA was included in the reaction). To dispersethe beads and limit diffusion during in situ capture, the expressionmixture was spread over the surface of a plain glass microscope slideand overlaid with a 18×18 mm cover glass (see Examples 25 and 26 formechanism and details of in situ capture). In situ capture was mediatedby a common N-terminal FLAG epitope tag present in all expressedproteins and the anti-FLAG PC-antibody on the beads. Expression wascarried out for 45 min at 42° C. in a humidified chamber withoutdisturbance or mixing. After expression, the microscope slide (and coverglass) “sandwich” was placed in a 50 mL polypropylene centrifuge tubeand sprayed at the seam with 400 μL of ice cold PBS and 10 mM EDTA (tubekept on crushed ice-water bath during this process; bead suspensioncollects at tube bottom). The bead suspension was recovered into theaforementioned Filtration Devices, filtration was performed and thefiltrate discarded. Beads were then washed 3×400 μL briefly with PBSthen 1×400 μL with PBS and 50% (v/v) glycerol. The washed bead pelletswere then resuspended to 1% beads (v/v) in PBS and 50% (v/v) glycerol.

Contact Photo-Transfer from Individually Resolved Beads:

Contact photo-transfer from individually resolved beads onto epoxyactivated glass microarray substrates (slides) (SuperEpoxy substrates,TeleChem International, Inc. ArrayIt™ Division, Sunnyvale, Calif.)overlaid with a cover glass was performed as described in Example 24with the following exceptions: 40 μL of the aforementioned 1% (v/v) beadsuspension was applied to the substrate and overlaid with a 18×18 mmsquare cover glass (coverslip). After contact photo-transfer, washingwas 3×30 sec with TBS-T only (cover glass removed) and substrates werenot dried. After washing, the substrates were further processed forantibody probing as described in the following paragraphs.

Antibody Probing and Detection:

Substrates were blocked for 10 min using excess 5% BSA (w/v) in TBS-T.Substrates were then probed to detect the common C-terminal VSV epitopetag present in all expressed proteins. To do so, a commercial anti-VSVantibody conjugated to the Cy3 fluorophore was used (clone P5D4;Sigma-Aldrich, St. Louis, Mo.). The antibody was diluted 1/100 from themanufacturer's stock in 5% BSA (w/v) in TBS-T. 100 μL of dilutedantibody probe was added to the substrate and overlaid with a 22×60 mmmicroscope cover glass. Binding was performed for 30 min at 37° C. in ahumidified chamber. The substrate was then washed 4× for 30 sec eachwith excess TBS-T followed by 4 brief washes in purified water. Thesubstrates were dried and then probed with a commercial anti-[mouse IgG]secondary antibody conjugated to the Alexa Fluor 488 fluorophore(Invitrogen Corporation, Carlsbad, Calif.) to detect the mouse anti-FLAGantibody present on the substrate in all contact photo-transfer spots(present regardless of the presence of cell-free expressed protein).Probing was done as above except that the antibody was diluted 1/1000and binding was overnight at +4° C. Substrates were washed and dried asabove and detection of the bound antibody probes was achieved by imagingthe dry microarray substrates on an ArrayWoRx^(e) BioChip fluorescencereader (Applied Precision, LLC, Issaquah, Wash.).

Results:

Results are shown in FIG. 37 as 2-color fluorescence image overlays foreach sample. Green represents signal from the anti-[mouse IgG] secondaryantibody conjugated to the Alexa Fluor 488 fluorophore which detects themouse anti-FLAG antibody present in all contact photo-transfer spots,regardless of the presence of expressed protein. The minus template(−Template) sample permutation was prepared in the same manner as theplus template (+Template) sample permutation except that only thetemplate DNA was omitted from the solid-phase bridge PCR reaction. Thered represents signal from the anti-VSV tag antibody conjugated to theCy3 fluorophore, which detects the common C-terminal VSV epitope tagpresent in both expressed proteins (p53 and GST A2). A yellow-orangecolor indicates binding of both fluorescent antibody probes. Arepresentative region is shown in FIG. 37, although approximately 100spots were analyzed for each sample. Results show that if template DNAwas omitted from the solid-phase bridge PCR reaction (−Template), nodetectible expressed protein is observed, but regardless, all spots aredetected (green) via the contact photo-transferred anti-FLAG captureantibody originally present on all beads. Expressed protein wasdetectible (red) only when template DNA was included in the solid-phasebridge PCR reaction (+Template). Importantly, only a fraction of thespots contain expressed protein, suggesting that 1 or a few of theoriginal template DNA molecules were amplified per bead.

Example 41 Effective Single Template Molecule Solid-Phase Bridge PCR:Multiplexed Cell-Free Expression with In Situ Protein Capture andOn-Bead Analysis by Flow Cytometry Preparing the Solid-Phase Bridge PCRTemplate DNA:

Performed as in Example 36.

Preparation of Agarose Beads Covalently Conjugated to PCR Primers Usedfor Solid-Phase Bridge PCR:

Performed as in Example 36.

Qualitative Analysis of Primer Attachment:

Performed as in Example 36.

First Round of Effective Single Template Molecule Solid-Phase BridgePCR:

Performed essentially as in Example 36, with slight modifications. Thefull protocol was as follows: 10 μL actual bead volume of the previouslyprepared Primer-Conjugated Agarose Beads was used per each sample, butfirst, each of the 10 μL of beads was washed separately in parallel,with heating. To do so, 50 μL each of the aforementioned 20% (v/v)Primer-Conjugated Agarose Bead suspension (10 μL actual bead volume) wasplaced into a 0.5 mL polypropylene thin-wall PCR tube. The beads werespun down briefly in a standard micro-centrifuge (just until reachesmaximum speed of ˜13,000 rpm corresponding to ˜16,000×g). As much of thefluid supernatant was removed as possible by manual pipetting, with thebeads nearly going to dryness. 40 μL each of TE-50 mM NaCl (10 mM Tris,pH 8.0, 1 mM EDTA, 50 mM NaCl) was added to the pellet, to bring thevolume back to the original 20% beads (v/v). The beads were brieflyvortex mixed then spun down and all fluid removed as described before.40 μL each of TE-50 mM NaCl was again added to the pellet as above andthe tube placed in a PCR machine (Mastercycler Personal; Eppendorf AG,Hamburg, Germany) at 95° C. for 10 min (lid temperature 105° C. and nomineral oil used) (beads were resuspended by brief gentle vortex mixingjust before and at 5 min of this step). After heating, the tube wasimmediately removed from the PCR machine, the beads diluted in 400 μL ofTE-50 mM NaCl and the bead suspension then transferred to a FiltrationDevice (see Example 36). Filtration was performed and the filtratediscarded. Beads were briefly washed 1×400 μL more with TE-50 mM NaClthen 1×400 μL with MBG-Water. Each set of beads was then resuspended in50 L MBG-Water and transferred to a 0.5 mL polypropylene thin-wall PCRtube. The beads were spun down briefly in a standard micro-centrifuge(just until reaches maximum speed of ˜13,000 rpm corresponding to˜16,000×g). As much of the fluid supernatant was removed as possible bymanual pipetting, with the beads nearly going to dryness.

Next, to pre-hybridize the template DNA to the washed Primer-ConjugatedAgarose Beads, each pellet was then resuspended in 5 μL of dilutedtemplate solution, which contained no soluble primers. The p53 and GSTA2 template mixture was prepared to 1 ng/μL as described in Example 36(except 75% GST A2 and 25% p53). This template mixture was furtherserially diluted to 0.05 ng/μL in a commercially available pre-mixed PCRreaction solution containing everything needed for PCR except templateDNA and primers (Platinum® PCR SuperMix High Fidelity; contains 22 U/mLcomplexed recombinant Taq DNA polymerase, Pyrococcus species GB-Dthermostable polymerase, Platinum® Taq Antibody, 66 mM Tris-SO₄ pH 8.9,19.8 mM (NH₄)₂SO₄, 2.4 mM MgSO₄, 220 μM dNTPs and stabilizers;Invitrogen Corporation, Carlsbad, Calif.; solution used without priordilution). This resulted in a ratio of 180 attomoles of template per μLof actual Primer-Conjugated Agarose Bead volume. With 1 μL ofPrimer-Conjugated Agarose Beads determined to contain approximately1,000 beads, 180 attomoles of template per μL of beads represents aratio of approximately 100,000 template molecules added per bead (beadsphysically enumerated under a microscope both in diluted droplets ofbead suspension and with suspensions in a hemacytometer cell countingchamber). A minus template negative control was also prepared. The beadsuspensions were only mixed manually by gentle stirring with a pipettetip.

The resultant bead suspensions, now containing added template but nosoluble (free) primers (only bead-bound primers), were then treated asfollows in a PCR machine (Mastercycler Personal; Eppendorf AG, Hamburg,Germany) (lid temperature 105° C. and no mineral oil used): 5 min 95° C.(denaturing), ramp down to 59° C. at a rate of 0.1° C./sec then hold 1hour at 59° C. (annealing/capture of template onto beads), 10 min 68° C.(fully extend any hybridized template-primer complexes once; no mixing).Immediately upon completion of the previous steps above, while the tubeswere still at 68° C., the tubes were immediately transferred from thePCR machine to a crushed ice water bath. 400 μL of ice cold MBG-Waterwas added to each tube, the suspensions transferred to fresh FiltrationDevices, filtration was immediately performed and the filtrate discarded(see Example 36). Using the same Filtration Devices, the beads werebriefly washed 2×400 μL with room temperature MBG-Water. Beads werefurther washed 2×400 μL for 2.5 min each with room temperature 0.1MNaOH, with constant vigorous vortex mixing, in order to strip off anyhybridized but non-covalently bound template DNA, leaving onlycovalently attached unused and extended primers on the beads. The beadswere then briefly washed 3×400 μL with 10×TE (100 mM Tris, pH 8.0, 10 mMEDTA), in order to neutralize the pH, followed by 3×400 μL withMBG-Water, in order to remove the components of the 10×TE which wouldinterfere with subsequent PCR.

Following the final filtration step on the bead samples, each washedbead pellet was resuspended in 100 μL of the commercial pre-mixed PCRsolution (Platinum® PCR SuperMix High Fidelity; Invitrogen Corporation,Carlsbad, Calif.) which was used at 92% strength (diluted withMBG-Water) and contains all necessary components for PCR except templateDNA and primers. The BODIPY-FL-dUTP labeling reagent was not used duringthe solid-phase bridge PCR. The suspensions were then recovered fromtheir Filtration Devices into fresh 0.5 mL polypropylene thin-wall PCRtubes and subjected to the following thermocycling in a PCR machine(Mastercycler Personal; Eppendorf AG, Hamburg, Germany) (lid temperature105° C. and no mineral oil used): An initial denaturing step of 94° C.for 2 min (once) (beads were briefly resuspended by gentle vortex mixingjust before and at the end of this step), and 40 cycles of 94° C. for 30sec (denature), 59° C. for 30 sec (anneal) and 68° C. for 2 min(extend); followed by a final extension step of 68° C. for 10 min(once).

400 μL of TE-50 mM NaCl-T (10 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl,0.01% v/v Tween-20) was added to each completed solid-phase bridge PCRreaction and the suspensions transferred to fresh 0.5 mL polypropylenePCR tubes. The beads were then spun down in a micro-centrifuge (justuntil reaches maximum speed of ˜13,000 rpm corresponding to ˜16,000×g)and the fluid supernatant carefully removed. The beads were washed 3×400μL more with TE-50 mM NaCl-T; resuspending by ˜5 sec vortex mixing thenspinning down and discarding the fluid supernatant as above. Followingthe final wash, as much of the fluid supernatant as possible was removedfrom the bead pellet by manual pipetting, with the beads going nearly todryness. The beads were lastly resuspended to 5% (v/v) using SP-PCRStorage Buffer (10 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl, all in 50%v/v glycerol). These intermediate beads could be stored at −20° C. andportions were subsequently used for a full second round of PCRthermocycling as described below.

Second Round of Solid-Phase Bridge PCR:

Performed as described in Example 36 and 37 except that a 2 μL portionof beads (actual bead volume) was used in 50 μL of the commerciallyavailable pre-mixed PCR reaction solution and without the BODIPY-FL-dUTPlabeling reagent (i.e. no BODIPY-FL-dUTP labeling at any stage).

Attaching the PC-Antibody to Beads Following Solid-Phase Bridge PCR:

Beads following the solid-phase bridge PCR reaction were used as thetest samples (−Template and +Template permutations) and, in addition,primer coated beads that were not subjected to the solid-phase bridgePCR reaction were used to generate the positive control. In all cases, 1μL actual bead volume per sample was washed briefly 3×400 μL with TE-T[10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 50 mM NaCl and 0.01% Tween-20(v/v)]. Unless otherwise noted, all washes and bead manipulations wereperformed in batch mode using 0.45 micron pore size, PVDF membrane,micro-centrifuge Filtration Devices to facilitate manipulation of thebeaded matrix (˜100 micron beads) and exchange the buffers (Ultrafree-MCDurapore Micro-centrifuge Filtration Devices, 400 μL capacity;Millipore, Billerica, Mass.). NeutrAvidin (tetrameric) was then attachedto the bead bound biotin-amine linker, in excess, by treatment with 200μL of a 0.5 μg/μL solution in TE-T for 20 min (note: biotin-amine linkerattached during previous preparation of Primer-Conjugated Agarose Beads;see Example 36). Beads were washed briefly 4×400 μL with TE-Saline (10mM Tris-HCl, pH 8.0, 1 mM EDTA and 200 mM NaCl.

The beads were next coated with a monoclonal mouse anti-FLAG tag captureantibody which was converted to photocleavable form by conjugation toPC-biotin. The antibody was additionally labeled with fluorescence toallow tracking of all beads, independent of the presence of expressedprotein (see later in this Example). To first create the fluorescentlylabeled PC-antibody (prepared in advance), 1 mg of antibody as suppliedby the manufacturer (Mouse Anti-FLAG M2 Antibody; Sigma-Aldrich, St.Louis, Mo.) was purified on a NAP-10 desalting column according to themanufacturer's instructions (Amersham Biosciences Corp., Piscataway,N.J.) against a 200 mM sodium bicarbonate and 200 mM NaCl buffer(nuclease-free reagents). 1 mL of the antibody elutate at 0.6 μg/μL waslabeled by adding 2 molar equivalents of a commercial Alexa Fluor 488TFP labeling reagent (Invitrogen Corporation, Carlsbad, Calif.) addedfrom a 12.5 mM stock in dimethylformamide (DMF). The reaction wascarried out for 30 min with gentle mixing. The antibody was then reactedwith 20 molar equivalents of AmberGen's PC-biotin-NHS labeling reagent(added from a 50 mM stock in anhydrous DMF) for 30 min with gentlemixing. The labeled antibody was then purified on a NAP-10 desaltingcolumn according to the manufacturer's instructions (AmershamBiosciences Corp., Piscataway, N.J.) against TBS. This preparedmonoclonal anti-FLAG PC-biotin fluorescent conjugate was then loadedonto the beads by treatment of the beads with 250 μL of 0.04 μg/μL inTE-Saline for 20 min. Beads were washed briefly 4×400 μL in TE-Salinefollowed by 2×400 μL in Molecular Biology Grade Water (MBG-Water).

Multiplexed Cell-Free Expression of the Beads and In Situ ProteinCapture:

For the test samples (−Template and +Template solid-phase bridge PCRpermutations), the 1 μL bead pellets were then resuspended in 25 μL ofthe E. coli based PureSystem cell-free expression mixture (mixtureprepared according to the manufacturer's instructions; Post GenomeInstitute Co., LTD., Japan) (no soluble DNA was included in the reactionexcept for the positive control sample; see below). To disperse thebeads and limit diffusion during in situ capture, the expression mixturewas spread over the surface of a plain glass microscope slide andoverlaid with a 18×18 mm cover glass (see Examples 25 and 26 formechanism and details of in situ capture). In situ capture was mediatedby a common N-terminal FLAG epitope tag present in all expressedproteins and the fluorescent anti-FLAG PC-antibody on the beads.Expression was carried out for 1 hr at 42° C. in a humidified chamberwithout disturbance or mixing. After expression, the microscope slide(and cover glass) “sandwich” was placed in a 50 mL polypropylenecentrifuge tube and sprayed at the seam with 400 μL of ice cold 5% BSA(w/v) in TBS-T (tube kept on crushed ice-water bath during this process;bead suspension collects at tube bottom). The bead suspension wasrecovered into the aforementioned Filtration Devices, filtration wasperformed and the filtrate discarded. Beads were then further washed2×400 μL briefly and 1×400 μL for 10 min in 5% BSA (w/v) in TBS-T.

The positive control beads, which were not previously subjected tosolid-phase bridge PCR, but were coated with PC-antibody as detailedearlier in this Example, were expressed similarly as above except:Expression was not performed on a glass microscope slide but in a tube(with mixing) by adding approximately 200 ng of the GST A2 solubletemplate DNA (see Example 36 for soluble GST A2 template DNA). Positivecontrol beads were then simply washed 1×400 μL with the ice cold 5% BSA(w/v) in TBS-T using the Filtration Devices then further washed 2×400 μLbriefly and 1×400 μL for 10 min in 5% BSA (w/v) in TBS-T.

Antibody Probing and Detection:

The beads were then probed to detect the common C-terminal VSV epitopetag present in all expressed proteins. To do so, a commercial anti-VSVantibody conjugated to the Cy3 fluorophore was used (clone PSD4;Sigma-Aldrich, St. Louis, Mo.). The antibody was diluted 1/250 from themanufacturer's stock in 5% BSA (w/v) in TBS-T. 250 μL of dilutedantibody probe was used to resuspend the washed bead pellets and bindingwas performed for 1 hr at 37° C. with mixing. Beads were washed briefly3×400 μL in TBS-T the 2×400 μL in PBS. Beads were then recovered fromthe Filtration Devices in 25 μL of PBS and analyzed in a BD FACSArray(BD Biosciences, San Jose, Calif.) flow cytometer.

Results:

Results are shown graphically in FIG. 38, whereby the X-axis is theintensity of the Cy3 labeled anti-VSV tag detection antibody and theY-axis the side-scatter (detection of all beads based on lightscattering). Based on the side-scatter, beads are identified in thelower left and lower right quadrants of each plot in FIG. 38, regardlessof fluorescence intensity. The minus template (−Template) samplepermutation was prepared in the same manner as the plus template(+Template) sample permutation except that only the template DNA wasomitted from the solid-phase bridge PCR reaction. The positive controlsample did not utilize solid-phase bridge PCR to generate theexpressible DNA, but instead used soluble PCR product for cell-freeexpression (did use capture on antibody coated beads) (for details see“Attaching the PC-Antibody to Beads Following Solid-Phase Bridge PCR”and “Multiplexed Cell-Free Expression of the Beads and In Situ ProteinCapture” sub-sections earlier in this Example). Beads were scoredpositive for detection with the Cy3 labeled anti-VSV tag antibody if thefluorescence intensity was sufficient such that they fell within thelower right quadrant of the plots in FIG. 38. The fluorescence intensitythreshold was set based on the positive control, such that the percentof positive beads in that sample was 20-fold greater than the percent ofpositive beads in the minus template negative control sample(−Template). In other words, the threshold was set to yield a 20:1signal to noise ratio for the positive control sample. Based on thesecriteria, 4% of the beads scored as positive in the plus template testsample (+Template) while 2% scored positive in the minus templatenegative control sample (−Template) for a 2:1 signal to noise ratio.Importantly, only a fraction of the beads contain expressed protein,suggesting that 1 or a few of the original template DNA molecules wereamplified per bead. This Example is similar to the previous Example 40except that here, final analysis directly on the beads via flowcytometry is demonstrated.

Example 42 Contact Photo-Transfer for Molecular Diagnostic Assays:Cell-Free Expression of the APC Gene Associated with Colorectal CancerFollowed by a Microarray Protein Truncation Test Based on FluorescenceAntibody Detection Preparation of a Photocleavable Antibody AffinityMatrix:

A polyclonal rabbit anti-HSV epitope tag capture antibody (BethylLaboratories, Montgomery, Tex.) was converted to photocleavable form byconjugation to photocleavable biotin (PC-biotin) as described in Example31. The resultant photocleavable antibody (PC-antibody) was then loadedonto a beaded affinity matrix. The following procedures, unlessotherwise noted, were performed in batch mode using Filtration Devicesto facilitate manipulation of the beaded matrix (˜100 micron beads),perform washes and otherwise exchange the buffers (FiltrationDevices=Ultrafree-MC Durapore Micro-centrifuge Filtration Devices, 400μL capacity, PVDF filtration membrane, 0.45 micron pore size; Millipore,Billerica, Mass. distributed by Sigma-Aldrich, St. Louis, Mo.). Unlessotherwise stated, all washes of the affinity matrix were by brief (˜5sec) vortex mixing in the Filtration Device, spinning down briefly in amicro-centrifuge (just until reaches maximum speed of ˜13,000 rpmcorresponding to ˜16,000×g) and discarding the filtrate. 4 μL packedvolume of NeutrAvidin biotin binding agarose beads (PierceBiotechnology, Inc., Rockford, Ill.) was washed 3×400 μL with TE-Saline(10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 200 mM NaCl). Beads were thenresuspended in 100 μL of 0.2 μg/μL PC-antibody in TE-Saline. Binding wasallowed to occur for 20 min with gentle mixing. Beads were then washed3×400 μL with TE-Saline and 1×400 μL with TE-Saline-T [10 mM Tris-HCl,pH 8.0, 1 mM EDTA, 200 mM NaCl, 0.1% (v/v) Tween-20]. Beads were thenrecovered from the Filtration Devices in 40 μL total by re-suspension inTE-Saline-T. The resultant 10% (v/v) PC-antibody beads were usedimmediately for capture of cell-free expressed proteins (see later inthis Example).

PCR Amplification of an APC Segment from Genomic DNA:

So-called segment 3 of either the wild-type or mutant APC gene wasamplified by PCR on genomic DNA from cell-lines as described in Example28 and reported by AmberGen in the scientific literature [Gite et al.(2003) Nat Biotechnol 21, 194-197]. The mutant APC gene contains atruncation mutation (nonsense mutation), in segment 3, that results in atruncated protein product upon translation.

Cell-Free Expression:

The PCR amplified APC segment 3 was expressed in a cell-free translationreaction as described by AmberGen in the scientific literature [Gite etal. (2003) Nat Biotechnol 21, 194-197].

Capture of the Cell-Free Expressed APC Segment on the PhotocleavableAntibody Affinity Matrix:

Following cell-free protein expression, 25 μL of the reaction mixturewas mixed with equal volume of Translation Dilution Buffer (TDB) [2×PBSpH 7.5, 0.4% (v/v) of a mammalian protease inhibitor cocktail (cocktailin DMSO, Sigma-Aldrich Corp., St. Louis, Mo.) and 20 mM EDTA added froma 500 nM pH 8.0 stock]. The samples were gently mixed for 10 min,supplemented with a final 0.1% (v/v) Tween-20 from a 10% (v/v) stock andmixed for an additional 5 min. The samples were clarified by spinning ona micro-centrifuge for 1 min (−˜13,000 rpm corresponding to ˜16,000×g)and subsequently collecting the supernatant. 10 μL of the aforementionedprepared 10% (v/v) PC-antibody beads was then added to each sample (1 μLpacked bead volume) and mixed for 30 min gently. Beads were then washed3×400 μL with PBS and 1×400 μL with 50% glycerol (v/v) in PBS using theaforementioned Filtration Devices. Beads were resuspended to 1% (v/v)with 50% glycerol (v/v) in PBS. Beads could be stored for at least 2days at −20° C. in this buffer.

Contact Photo-Transfer from Individually Resolved Beads:

Contact photo-transfer from individually resolved beads onto epoxyactivated glass microarray substrates (slides) (SuperEpoxy substrates,TeleChem International, Inc. ArrayIt™ Division, Sunnyvale, Calif.)overlaid with a cover glass was performed as described in Example 24with the following exceptions: 50 μL of the aforementioned 1% (v/v) beadsuspension was applied to the substrate and overlaid with a 18×18 mmsquare cover glass (coverslip). After contact photo-transfer, TBS-Twashes were 2×2 min (cover glass removed). After washing and drying, thesubstrates were further processed for antibody probing as described inthe following paragraphs.

Antibody Probing and Detection:

Substrates were blocked for 10 min using excess 5% BSA (w/v) in TBS-T.

Substrates were then probed to detect the N-terminal VSV epitope tag(YTDIEMNRLGK [SEQ NO. 33]), present in all APC segment 3 proteinproducts, as well as a C-terminal p53-derived epitope tag (TFSDLHKLL[SEQ NO. 34]), present only in non-truncated (full-length) APC segment 3protein products. To do so, a commercial anti-VSV antibody conjugated tothe Cy3 fluorophore (clone P5D4; Sigma-Aldrich, St. Louis, Mo.) and anin-house prepared anti-p53 antibody conjugated to the Cy5 fluorophore(Example 26) were used. Both antibodies were added to the same solutionfor dual simultaneous probing. The anti-VSV-Cy3 antibody was diluted1/500 and the anti-p53-Cy5 antibody 1/50 with 5% BSA (w/v) in TBS-T. 100μL of the antibody probing solution was added to the substrate andoverlaid with a 22×60 mm microscope cover glass. Binding was performedfor 30 min at 37° C. in a humidified chamber. The substrate was thenwashed 3× for 2 min each with excess TBS-T followed by 4 brief washes inpurified water. The substrates were dried and detection of the boundantibody probes was achieved by imaging the dry microarray substrates onan ArrayWoRx^(e) BioChip fluorescence reader (Applied Precision, LLC,Issaquah, Wash.).

Results:

Results are shown in FIG. 39A as 2-color fluorescence image overlays foreach sample permutation. Green corresponds to signal from theanti-VSV-Cy3 N-terminal epitope tag antibody and red the anti-p53-Cy5C-terminal epitope tag antibody. The yellow-orange color indicates thepresence of both the green and red signals in the 2-color fluorescenceimage overlay. The minus DNA (−DNA) sample permutation is identical tothe other sample permutations except that expressible APC DNA wasomitted from the cell-free translation reaction. Qualitatively, asexpected, the APC wild-type (APC WT) shows signals for both the N- andC-terminal epitope tags (green and red), while the APC mutant (i.e.truncated) shows only the N-terminal signal (green only).

For more precise data interpretation, the non-overlaid raw fluorescencegrayscale images were quantified by computer-assisted image analysisusing the ImageQuant software package (Molecular Dynamics; AmershamBiosciences Corp., Piscataway, N.J.). Average fluorescence intensitiesfor each spot (henceforth referred to as “spot intensity”) weredetermined in both the green and red fluorescence channels (i.e. averagefluorescence intensity over the entire area of a given individual spot).More than 300 spots were quantified for each sample permutation (exceptthe −DNA negative control where no discrete spots were discernable).Using these data, without background subtraction, the C-terminal toN-terminal ratios (so-called C:N ratio) for each spot were calculated(i.e. ratio of red to green spot intensities) and averaged. The datawere uniformly normalized to such that the C:N ratio of the APCwild-type (APC WT) was set to 100%. As shown graphically in FIG. 39B,the average C:N ratio of the APC WT was 100±12% and the APC mutant 5±1%,a 20-fold difference. N-terminal signal to noise ratios were an average186:1 and 246:1 for the APC WT and APC mutant respectively. C-terminalsignal to noise ratios were an average 21:1 and 1:1 for the APC WT andAPC mutant respectively.

Example 43 Solid-Phase Bridge PCR on the APC Gene Associated withColorectal Cancer: Cell-free Expression and Contact Photo-TransferFollowed by a Microarray Protein Truncation Test Based on FluorescenceAntibody Detection

This Example is similar to the previous Example 42, except thatsolid-phase bridge PCR was used to generate the expressible APC DNA. Thebeads carrying the APC solid-phase bridge PCR product (amplicon) werecoated with a photocleavable antibody (PC-antibody) for downstreamprotein capture, the beads then used directly in a cell-free expressionreaction and translated APC proteins were captured onto the same beadsvia the PC-antibody. Contact photo-transfer was then performed tofabricate random microarrays and the resultant spots were then probedwith fluorescent antibodies against N- and C-terminal epitope tags,which can allow detection of truncated APC protein products as shownpreviously in Example 42.

The integration of solid-phase bridge PCR into this process affordsseveral advantages, including but not limited to: a) The ability tomultiplex, in a single solid-phase bridge PCR reaction, theamplification of various APC segments (e.g. different exons or fragmentsthere of) or b) the ability to perform amplification of 1 or a few APCtemplate molecules per each bead, in order to facilitate for example,high sensitivity detection of a few mutant APC template molecules in thepresence of an excess of wild-type APC template molecules, based on asingle solid-phase bridge PCR reaction. These processes are not intendedto be limited to the APC gene, but are applicable to other nucleic acidsequences.

Preparing the Solid-Phase Bridge PCR Template DNA:

Note: All buffers and reagents used throughout this entire Example,unless otherwise noted, were minimally DNAse, RNAse and protease free,referred to as Molecular Biology Grade (MBG), including the water,referred to as MBG-Water.

Soluble wild-type (WT) and mutant (nonsense, i.e. truncation) versionsof APC segment 3, of approximately 1.7 kb in size, were produced fromcell-line genomic DNA as described in Example 28 and as described byAmberGen in the scientific literature [Gite et al. (2003) Nat Biotechnol21, 194-197]. This DNA product was further amplified using standardsolution-phase PCR practices using the following APC-specific primer set(APC-specific hybridizing sequences are bracketed and the remainingsequences are a portion of those necessary for efficient cell-freeexpression and incorporation of epitope tags. The remaining sequencesneeded for efficient cell-free expression and incorporation of epitopetags are introduced via the solid-phase bridge PCR primers used later):

Forward Primer: [SEQ NO. 35]5′ATgAACCgCCTgggCAAgggAggAggAggACAgCCTgAACTCgCTCCAgAggATCCggAAgAT[CAggAAgCAgATTCTgCTAAT]3′ Reverse Primer: [SEQ NO. 36]5′TTACAgCAgCTTgTgCAggTCgCTgAAggT[gggTgTCTgAgCACCAC TTTT]3′

This resulted in a 321 bp APC product, from within the so-called APCmutation cluster region (MCR), that was used (without purification) asthe template for solid-phase bridge PCR. The 321 bp product covers APCcodons 1,294-1,369 with the truncation mutation located at site 1,338(CAg→TAg).

Preparation of Agarose Beads Covalently Conjugated to PCR Primers Usedfor Solid-Phase Bridge PCR:

Performed as in Example 36 except that 25 μM final concentration of eachprimer was used and 90 μL of the primer solution (containing 25 μM eachprimer) was added to 50 μL packed bead volume for attachment (12 μMfinal of the Biotin-Amine Linker was used as in Example 36). Sequencesof the 5′ primary amine modified (6 carbon spacer) primers used forattachment to the beads were as follows:

Forward Primer: [SEQ NO. 37]5′[Amine]TAATACgACTCACTATAgggAgAggAggTATATCAATgTACACCgACATCgAgATgAACCgCCTgggCAAgggAggAggAggA3′ Reverse Primer: [SEQ NO.38] 5′[Amine]TTACAgCAgCTTgTgCAggTCgCTgAAggTgggTgTCTgAg CACCACTTTT3′

Based on the primers used, the final APC solid-phase bridge PCR product(on the beads) would contain additional untranslated sequences forefficient cell-free expression as well as sequences for epitope tags.Specifically, an N-terminal VSV epitope tag (YTDIEMNRLGK [SEQ NO. 39])for detection, followed by a 4 glycine spacer, an N-terminal HSV epitopetag (QPELAPEDPED [SEQ NO. 40]) for protein capture and a C-terminalp53-derived epitope tag (TFSDLHKLL [SEQ NO. 41]) for detection. The N-and C-terminal epitope tags flank the APC gene fragment insert.

Qualitative Analysis of Primer Attachment:

Performed as in Example 36.

First Round of Solid-Phase Bridge PCR:

Performed essentially as in Example 36, with slight modifications. Thefull protocol was as follows: 5 μL actual bead volume of the previouslyprepared Primer-Conjugated Agarose Beads was used per each sample, butfirst, each of the 5 μL of beads was washed separately in parallel, withheating. To do so, 25 μL each of the aforementioned 20% (v/v)Primer-Conjugated Agarose Bead suspension (5 μL actual bead volume) wasplaced into a 0.5 mL polypropylene thin-wall PCR tube. The beads werespun down briefly in a standard micro-centrifuge (just until reachesmaximum speed of ˜13,000 rpm corresponding to ˜16,000×g). As much of thefluid supernatant was removed as possible by manual pipetting, with thebeads nearly going to dryness. 20 μL each of TE-50 mM NaCl (10 mM Tris,pH 8.0, 1 mM EDTA, 50 mM NaCl) was added to the pellet, to bring thevolume back to the original 20% beads (v/v). The beads were brieflyvortex mixed then spun down and all fluid removed as described before.20 μL each of TE-50 mM NaCl was again added to the pellet as above andthe tube placed in a PCR machine (Mastercycler Personal; Eppendorf AG,Hamburg, Germany) at 95° C. for 10 min (lid temperature 105° C. and nomineral oil used) (beads were resuspended by brief gentle vortex mixingjust before and at 5 min of this step). After heating, the tube wasimmediately removed from the PCR machine, the beads diluted in 400 μL ofTE-50 mM NaCl and the bead suspension then transferred to a FiltrationDevice (see Example 36). Filtration was performed and the filtratediscarded. Beads were briefly washed 1×400 μL more with TE-50 mM NaClthen 1×400 μL with MBG-Water. Each set of beads was then resuspended in50 μL MBG-Water and transferred to a 0.5 mL polypropylene thin-wall PCRtube. The beads were spun down briefly in a standard micro-centrifuge(just until reaches maximum speed of ˜13,000 rpm corresponding to˜16,000×g). As much of the fluid supernatant was removed as possible bymanual pipetting, with the beads nearly going to dryness.

Next, to pre-hybridize the template DNA to the washed Primer-ConjugatedAgarose Beads, each pellet was then resuspended in 2.5 μL of dilutedtemplate solution, which contained no soluble primers. The APC templatewas prepared to 0.16 ng/μL at a 75% wild-type and 25% mutant mixture.The template was prepared in a commercially available pre-mixed PCRreaction solution containing everything needed for PCR except templateDNA and primers (Platinum® PCR SuperMix High Fidelity; contains 22 U/mLcomplexed recombinant Taq DNA polymerase, Pyrococcus species GB-Dthermostable polymerase, Platinum® Taq Antibody, 66 mM Tris-SO₄ pH 8.9,19.8 mM (NH₄)₂SO₄, 2.4 mM MgSO₄, 220 μM dNTPs and stabilizers;Invitrogen Corporation, Carlsbad, Calif.; solution used without priordilution). This resulted in a ratio of 400 attomoles of template per μLof actual Primer-Conjugated Agarose Bead volume. With 1 μL ofPrimer-Conjugated Agarose Beads determined to contain approximately1,000 beads, 400 attomoles of template per μL of beads represents aratio of roughly 200,000 template molecules added per bead (beadsphysically enumerated under a microscope both in diluted droplets ofbead suspension and with suspensions in a hemacytometer cell countingchamber). A minus template negative control was also prepared. The beadsuspensions were only mixed manually by gentle stirring with a pipettetip.

The resultant bead suspensions, now containing added template but nosoluble (free) primers (only bead-bound primers), were then treated asfollows in a PCR machine (Mastercycler Personal; Eppendorf AG, Hamburg,Germany) (lid temperature 105° C. and no mineral oil used): 5 min 95° C.(denaturing), ramp down to 55° C. at a rate of 0.1° C./sec then hold 1hour at 55° C. (annealing/capture of template onto beads), 10 min 68° C.(fully extend any hybridized template-primer complexes once; no mixing).Immediately upon completion of the previous steps above, while the tubeswere still at 68° C., the tubes were immediately transferred from thePCR machine to a crushed ice water bath. 400 μL of ice cold MBG-Waterwas added to each tube, the suspensions transferred to fresh FiltrationDevices, filtration was immediately performed and the filtrate discarded(see Example 36). Using the same Filtration Devices, the beads werebriefly washed 2×400 μL with room temperature MBG-Water. Beads werefurther washed 2×400 μL for 2.5 min each with room temperature 0.1MNaOH, with constant vigorous vortex mixing, in order to strip off anyhybridized but non-covalently bound template DNA, leaving onlycovalently attached unused and extended primers on the beads. The beadswere then briefly washed 3×400 μL with 10×TE (100 mM Tris, pH 8.0, 10 mMEDTA), in order to neutralize the pH, followed by 3×400 μL withMBG-Water, in order to remove the components of the 10×TE which wouldinterfere with subsequent PCR.

Following the final filtration step on the bead samples, each washedbead pellet was resuspended in 100 μL of the commercial pre-mixed PCRsolution (Platinum® PCR SuperMix High Fidelity; Invitrogen Corporation,Carlsbad, Calif.) which was used at 92% strength (diluted withMBG-Water) and contains all necessary components for PCR except templateDNA and primers. The BODIPY-FL-dUTP labeling reagent was not used in thesolid-phase bridge PCR reaction. The suspensions were then recoveredfrom their Filtration Devices into fresh 0.5 mL polypropylene thin-wallPCR tubes and subjected to the following thermocycling in a PCR machine(Mastercycler Personal; Eppendorf AG, Hamburg, Germany) (lid temperature105° C. and no mineral oil used): An initial denaturing step of 94° C.for 2 min (once) (beads were briefly resuspended by gentle vortex mixingjust before and at the end of this step), and 40 cycles of 94° C. for 30sec (denature), 59° C. for 30 sec (anneal) and 68° C. for 2 min(extend); followed by a final extension step of 68° C. for 10 min(once).

400 μL of TE-50 mM NaCl-T (10 nM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl,0.01% v/v Tween-20) was added to each completed solid-phase bridge PCRreaction and the suspensions transferred to the aforementionedFiltration Devices for washing. The beads were washed 3×400 μL with theTE-50 mM NaCl-T then 3×400 μL with MBG-Water; resuspending by ˜5 secvortex mixing then performing filtration and discarding the filtrate.The washed beads were immediately used for a full second round of PCRthermocycling as described below.

Second Round of Solid-Phase Bridge PCR:

Performed as described in Example 37 except that the washed bead pelletsfrom above, still in their Filtration Devices, were directly resuspendedin the solid-phase bridge PCR reaction mixture and transferred to 0.5 mLthin-walled polypropylene PCR tubes for thermocycling.

Attaching the PC-Antibody to Beads Following Solid-Phase Bridge PCR:

Following completion of the solid-phase bridge PCR, NeutrAvidin and thena photocleavable antibody (PC-antibody) were loaded onto the beads. Thiswas performed as described in Example 40 with the following exceptions.2 μL packed bead volume per sample was used and was pre-washed only2×400 μL with TE-Saline (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 200 mM NaCl)instead of 3×. NeutrAvidin was used at 0.2 μg/μL instead of 0.5 μg/μL.Instead of coating with an anti-FLAG capture antibody, the beads werecoated with a polyclonal rabbit anti-HSV tag capture antibody (BethylLaboratories, Montgomery, Tex.) which was converted to photocleavableform by conjugation to PC-biotin. Creation of the photocleavableantibody (PC-antibody) was performed as described in Example 31. ThePC-antibody as added at 0.1 μg/μL (100 μL/sample) and binding wasallowed to occur for 30 min. In addition to the washes described inExample 40, the beads were finally washed additionally 1×400 μL with 50%glycerol in TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) and resuspended to 4%(v/v) beads in the same buffer. Beads were stored overnight at −20° C.prior to continuation of the procedure as described below.

Multiplexed Cell-Free Expression of the Beads and In Situ ProteinCapture:

Performed as in Example 40 with the following exceptions: The storedbeads from above (1 μL packed volume) were first pre-washed, prior toexpression, 2×400 μL in MBG-Water using the Filtration Devices (seeExample 40 for washing with Filtration Devices). The expression reactionwas carried out for 60 min at 37° C. After expression, the beads wereinitially recovered in 400 μL of ice cold PBS and 10 mM EDTA as inExample 40 except the buffer was additionally supplemented with 0.05%(v/v) Tween-20. The washed bead pellets were ultimately resuspended to1.25% beads (v/v) in PBS and 50% (v/v) glycerol prior to performingcontact photo-transfer, instead of 1% (v/v) beads.

Contact Photo-Transfer from Individually Resolved Beads:

Performed as in Example 40 except that the 1.25% (v/v) beads suspensionwas used for contact photo-transfer.

Antibody Probing and Detection:

Performed as in Example 42 except: The anti-VSV-Cy3 antibody was diluted1/100 and the anti-p53-Cy5 antibody 1/100, instead of 1/500 and 1/50respectively. After antibody probing, the TBS-T washes were for 30 seceach instead of 2 min each.

Results:

Results are shown in FIG. 40 as non-overlaid fluorescence grayscaleimages of the same microarray region for each sample permutation.Qualitatively, it is observed that both the C-terminal p53-derived andthe N-terminal VSV epitope tags are detectible in each contactphoto-transferred spot, only in the sample permutation where templateDNA was included in the solid-phase bridge PCR reaction (+Template). Ifonly the template DNA was omitted at the level of the solid-phase bridgePCR reaction, neither epitope tag is detected at the protein level onthe contact photo-transfer microarray (−Template). The wild-type (WT)and mutant (truncated) APC protein products were not measurablysegregated on different beads (spots), hence the detection of bothepitope tags in each spot. This data therefore suggests significantlymore than 1 or few original template DNA molecules were amplified perbead during solid-phase bridge PCR, with this particular experimentalsetup. This APC experimental system is comprised of a different templatespecies and a different primer pair compared to the p53-GST A2solid-phase bridge PCR system used in Examples 36-41, and additionallyused altered solid-phase bridge PCR conditions relative to the p53-GSTA2 system (e.g. longer primers with different T_(m) values and lowertemperature during initial template capture step). Therefore, the addedtemplate:bead ratio needed to achieve amplification of 1 or few originaltemplate DNA molecules per bead is significantly different, as evidencedin the subsequent Example 44.

The images were quantified by computer-assisted image analysis using theImageQuant software package (Molecular Dynamics; Amersham BiosciencesCorp., Piscataway, N.J.). Average fluorescence intensities for each spot(henceforth referred to as “spot intensity”) were determined in bothfluorescence channels (i.e. average fluorescence intensity over theentire area of a given individual spot). More than 300 spots werequantified in the plus template (+Template) sample permutation. Averagesignal to noise ratios were 3±1:1 and 179±29:1 for the C-terminalp53-derived and the N-terminal VSV epitope tags respectively.

Example 44 Effective Single Template Molecule Solid-Phase Bridge PCR onthe APC Gene Associated with Colorectal Cancer: Validation of EffectiveAmplification of Single Template Molecules per Bead Using 2 TemplateSpecies and a Single-Base Extension Reaction as the Ultimate AssayPreparing the Solid-Phase Bridge PCR Template DNA:

Note: All buffers and reagents used throughout this entire Example,unless otherwise noted, were minimally DNAse, RNAse and protease free,referred to as Molecular Biology Grade (MBG), including the water,referred to as MBG-Water.

Templates used for solid-phase bridge PCR were generated via initialstandard solution-phase PCR. A segment of Exon 15 of the human APC gene(codons 1,294-1,369) (see GeneBank M74088 for full APC open readingframe) was amplified using solution-phase PCR with gene-specificprimers, essentially via standard PCR and molecular biology practices.For the solution-phase PCR, genomic DNA from the HeLa cell line was usedas the wild-type template and genomic DNA from the SW480 colorectalcancer cell line as mutant template. The SW480 cell line possesses anonsense mutation (CAg→TAg) in the APC gene at codon 1,338 resulting ina truncated gene product (protein) upon expression. Isolation of genomicDNA from cultured cells and solution-phase PCR amplification of thehuman APC gene was essentially performed as reported by AmberGen in thescientific literature [Gite et al. (2003) Nat Biotechnol 21, 194-197]with the following exceptions: PCR primers used in this Example arelisted below this paragraph. In the primers below, the bracketedsequences indicate the gene-specific hybridization regions, while theremaining sequences are non-hybridizing regions which act as commonuniversal sequences, flanking the gene fragment, to which the subsequentsolid-phase bridge PCR primers are directed (the non-hybridizing regionsalso correspond to partial elements needed for later cell-free proteinexpression as well as epitope tag detection; the remaining portion ofthese elements are introduced via the solid-phase bridge PCR primersdetailed later in this Example). 0.1 μM of each primer was used and thePCR system was a commercially available pre-mixed PCR reaction solutioncontaining everything needed for PCR except template DNA and primers(Platinum® PCR SuperMix High Fidelity; contains 22 U/mL complexedrecombinant Taq DNA polymerase, Pyrococcus species GB-D thermostablepolymerase, Platinum® Taq Antibody, 66 mM Tris-SO₄ pH 8.9, 19.8 mM(NH₄)₂SO₄, 2.4 mM MgSO₄, 220 μM dNTPs and stabilizers; InvitrogenCorporation, Carlsbad, Calif.; solution used at 90% strength). Thefollowing thermocycling steps were used: Initially 94° C. 2 min (once)and then 35 cycles of 94° C. 30 s, 60° C. 30 s and 68° C. 30 s, followedby a final 68° C. 5 min (once).

Solution-Phase PCR APC Forward Primer: [SEQ NO. 42]5′ATgAACCgCCTgggCAAgggAggAggAggACAgCCTgAACTCgCTCCAgAggATCCggAAgAT[CAggAAgCAgATTCTgCTAAT]3′ Solution-Phase PCR APC ReversePrimer: [SEQ NO. 43] 5′TTACAgCAgCTTgTgCAggTCgCTgAAggT[gggTgTCTgAgCACCACTTTT]3′Following the solution-phase PCR, the products were analyzed by standardagarose gel electrophoresis and ethidium bromide staining to ensure asingle band was produced and of the correct molecular weight. Based onthe primers used, the PCR products are 321 bp. The PCR products werethen purified by agarose gel electrophoresis. These purified PCRproducts subsequently served as template DNA for the solid-phase bridgePCR reactions described later in this Example.

Preparation of Agarose Beads Covalently Conjugated to PCR Primers Usedfor Solid-Phase Bridge PCR:

Performed as in Example 36 with the following exceptions: PCR primersused in this Example are listed below this paragraph. In the primersbelow, the bracketed sequences indicate the template-specifichybridization regions, while the remaining sequences are non-hybridizingregions which correspond to the remaining portions of the elementsneeded for later cell-free protein expression as well as epitope tagdetection (the initial portion of these elements was introduced duringthe template preparation earlier in this Example). During conjugation tothe beads, concentration of each primer was 29 μM instead of 125 μM.

Solid-Phase Bridge PCR APC Forward Primer: [SEQ NO. 44]5′[Amine]TAATACgACTCACTATAgggAgAggAggTATATCAATgTACACCgACATCgAg[ATgAACCgCCTgggCAAgggAggAggAggA]3′ Solid-Phase Bridge PCRAPC Reverse Primer: [SEQ NO. 45]5′[Amine]TTTTTTTTTTTTTTTTTTTTATTATCCTCCTCCTgCgTAgTCTggTACgTCgTATgggTA[CAgCAgCTTgTgCAggTCgCTgAAggTg g]3′

Qualitative Analysis of Primer Attachment:

Performed as in Example 36.

First Round of Effective Single Template Molecule Solid-Phase BridgePCR:

Performed essentially as in Example 36, with slight modifications. Thefull protocol was as follows: 10 μL actual bead volume of the previouslyprepared Primer-Conjugated Agarose Beads was used per each sample, butfirst, each of the 10 μL of beads was washed separately in parallel,with heating. To do so, 50 μL each of the aforementioned 20% (v/v)Primer-Conjugated Agarose Bead suspension (10 μL actual bead volume) wasplaced into a 0.5 mL polypropylene thin-wall PCR tube. The beads werespun down briefly in a standard micro-centrifuge (just until reachesmaximum speed of ˜13,000 rpm corresponding to ˜16,000×g). As much of thefluid supernatant was removed as possible by manual pipetting, with thebeads nearly going to dryness. 40 μL each of TE-50 mM NaCl (10 mM Tris,pH 8.0, 1 mM EDTA, 50 mM NaCl) was added to the pellet, to bring thevolume back to the original 20% beads (v/v). The beads were brieflyvortex mixed then spun down and all fluid removed as described before.40 μL each of TE-50 mM NaCl was again added to the pellet as above andthe tube placed in a PCR machine (Mastercycler Personal; Eppendorf AG,Hamburg, Germany) at 95° C. for 10 min (lid temperature 105° C. and nomineral oil used) (beads were resuspended by brief gentle vortex mixingjust before and at 5 min of this step). After heating, the tube wasimmediately removed from the PCR machine, the beads diluted in 400 μL ofTE-50 mM NaCl and the bead suspension then transferred to a FiltrationDevice (see Example 36). Filtration was performed and the filtratediscarded. Beads were briefly washed 1×400 μL more with TE-50 mM NaClthen 1×400 μL with MBG-Water. Each set of beads was then resuspended in50 μL MBG-Water and transferred to a 0.5 mL polypropylene thin-wall PCRtube. The beads were spun down briefly in a standard micro-centrifuge(just until reaches maximum speed of ˜13,000 rpm corresponding to˜16,000×g). As much of the fluid supernatant was removed as possible bymanual pipetting, with the beads nearly going to dryness.

Next, to pre-hybridize the template DNA to the washed Primer-ConjugatedAgarose Beads, each pellet was then resuspended in 5 μL of dilutedtemplate solution, which contained no soluble primers. Theaforementioned APC template, prepared as described in this Example, wasdiluted to 8×10⁻⁷ ng/μL, mixed at a ratio of 50% wild-type (WT) and 50%mutant APC. The template was prepared in a commercially availablepre-mixed PCR reaction solution containing everything needed for PCRexcept template DNA and primers (Platinum® PCR SuperMix High Fidelity;contains 22 U/mL complexed recombinant Taq DNA polymerase, Pyrococcusspecies GB-D thermostable polymerase, Platinum® Taq Antibody, 66 mMTris-SO₄ pH 8.9, 19.8 mM (NH₄)₂SO₄, 2.4 mM MgSO₄, 220 μM dNTPs andstabilizers; Invitrogen Corporation, Carlsbad, Calif.; solution usedwithout prior dilution). This resulted in a ratio of 0.002 attomoles oftemplate per μL of actual Primer-Conjugated Agarose Bead volume. With 1μL of Primer-Conjugated Agarose Beads determined to containapproximately 1,000 beads, 0.002 attomoles of template per μL of beadsrepresents a ratio of approximately 1 template molecule added per bead(beads physically enumerated under a microscope both in diluted dropletsof bead suspension and with suspensions in a hemacytometer cell countingchamber). A minus template negative control was also prepared. The beadsuspensions were only mixed manually by gentle stirring with a pipettetip.

The resultant bead suspensions, now containing added template but nosoluble (free) primers (only bead-bound primers), were then treated asfollows in a PCR machine (Mastercycler Personal; Eppendorf AG, Hamburg,Germany) (lid temperature 105° C. and no mineral oil used): 5 min 95° C.(denaturing), ramp down to 55° C. at a rate of 0.1° C./sec then hold 1hour at 55° C. (annealing/capture of template onto beads), 10 min 68° C.(fully extend any hybridized template-primer complexes once; no mixing).Immediately upon completion of the previous steps above, while the tubeswere still at 68° C., the tubes were immediately transferred from thePCR machine to a crushed ice water bath. 400 μL of ice cold MBG-Waterwas added to each tube, the suspensions transferred to fresh FiltrationDevices, filtration was immediately performed and the filtrate discarded(see Example 36). Using the same Filtration Devices, the beads werebriefly washed 2×400 μL with room temperature MBG-Water. Beads werefurther washed 2×400 μL for 2.5 min each with room temperature 0.1MNaOH, with constant vigorous vortex mixing, in order to strip off anyhybridized but non-covalently bound template DNA, leaving onlycovalently attached unused and extended primers on the beads. The beadswere then briefly washed 3×400 μL with 10×TE (100 mM Tris, pH 8.0, 10 mMEDTA), in order to neutralize the pH, followed by 3×400 μL withMBG-Water, in order to remove the components of the 10×TE which wouldinterfere with subsequent PCR.

Following the final filtration step on the bead samples, each washedbead pellet was resuspended in 100 μL of the commercial pre-mixed PCRsolution (Platinum® PCR SuperMix High Fidelity; Invitrogen Corporation,Carlsbad, Calif.) which was used at 92% strength (diluted withMBG-Water) and contains all necessary components for PCR except templateDNA and primers. The solid-phase bridge PCR reaction was furthersupplemented with 0.15 U/μL final of additional PlatinumTaq DNAPolymerase High Fidelity added from a 5 U/μL manufacturer's stock(Invitrogen Corporation, Carlsbad, Calif.). The BODIPY-FL-dUTP labelingreagent was not used in the solid-phase bridge PCR reaction. Thesuspensions were then recovered from their Filtration Devices into fresh0.5 mL polypropylene thin-wall PCR tubes and subjected to the followingthermocycling in a PCR machine (Mastercycler Personal; Eppendorf AG,Hamburg, Germany) (lid temperature 105° C. and no mineral oil used): Aninitial denaturing step of 94° C. for 2 min (once) (beads were brieflyresuspended by gentle vortex mixing just before this step), and 35cycles of 94° C. for 1 min (denature), 68° C. for 2 min (anneal andextend); followed by a final extension step of 68° C. for 10 min (once).

400 μL of TE-50 mM NaCl (10 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl) wasadded to each completed solid-phase bridge PCR reaction and thesuspensions transferred to Filtration Devices (Ultrafree-MC DuraporeMicro-centrifuge Filtration Devices, 400 μL capacity; Millipore,Billerica, Mass.). Filtration was performed in a micro-centrifuge (justuntil reaches maximum speed of ˜13,000 rpm corresponding to ˜16,000×g)and the filtrate discarded. The beads were washed 3×400 μL more withTE-50 mM NaCl; resuspending by ˜5 sec vortex mixing then performingfiltration and discarding the filtrate as above. Beads were usedimmediately for a full second round of PCR thermocycling as describedbelow.

Second Round of Solid-Phase Bridge PCR:

Performed as described in Example 36 and 37 except that a 10 μL portionof beads (actual bead volume) was used in 100 μL of the commerciallyavailable pre-mixed PCR reaction solution and without the BODIPY-FL-dUTPlabeling reagent (i.e. no BODIPY-FL-dUTP labeling at any stage).Furthermore, the solid-phase bridge PCR reaction was furthersupplemented with 0.15 U/μL final of additional PlatinumTaq DNAPolymerase High Fidelity added from a 5 U/μL manufacturer's stock(Invitrogen Corporation, Carlsbad, Calif.). Thermocycling was performedas above in this Example.

400 μL of TE-50 mM NaCl (10 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl) wasadded to each completed solid-phase bridge PCR reaction and thesuspensions transferred to fresh Filtration Devices (Ultrafree-MCDurapore Micro-centrifuge Filtration Devices, 400 μL capacity;Millipore, Billerica, Mass.). Filtration was performed and the filtratediscarded. The beads were washed briefly 3×400 μL more with TE-50 mMNaCl. Beads were additionally washed 2×400 μL briefly with MBG-Waterbefore processing further as described below in this Example.

Single-Base Extension Reaction to Detect Mutant and Wild-Type APC:

A single-base extension (SBE) reaction was performed with fluorescentdideoxynucleotide triphosphates (ddNTPs) in order to distinguish themutant (TAg at codon 1,338) from the wild-type (CAg at codon 1,338) APCamplicon on the solid-phase bridge PCR beads. Hence, a hybridizationoligonucleotide probe was used in the SBE reaction which hybridizes upto (just before), but not overlapping with, the mutation site on thesolid-phase bridge PCR amplicon (product). Cy5 labeled ddUTP and Cy3labeled ddCTP were used in the extension reaction to detect the mutantand wild-type APC respectively.

First however, several prior measures were taken to eliminate backgroundin the SBE reaction. The beads were first treated with Exonuclease I(New England Biolabs, Inc., Ipswich, Mass.) to remove any unusedprimers. To do so, following the final washes as detailed above, beadswere resuspended in 30 μL of 1× reaction buffer (67 mM Glycine-KOH, 6.7mM MgCl2, 10 mM 2-Mercaptoethanol, pH 9.5@25° C.) containing 0.7 U/μLfinal concentration of Exonuclease I. The reaction was incubated for 1hr at 37° C. and the enzyme then heat inactivated for 10 min at 90° C.Using the aforementioned Filtration Devices, the beads were then washed2×400 μL in 0.1 N NaOH for 3 min each with gentle mixing. Beads werethen washed briefly 3×400 μL in 10×TE (100 mM Tris, pH 8.0, 10 mM EDTA)then 3×400 μL in TE-50 mM NaCl.

Next, the solid-phase bridge PCR amplicon on the beads was pre-cappedusing unlabeled ddNTP terminators. To do so, the beads were placed in 80μL of 1× ThermoSequenase Reaction Buffer (150 mM Tris-HCl, pH 9.5, 67 mMMgCl₂) with 25 μM of each of the 4 ddNTPs, and 0.5 U/μL of theThermoSequenase DNA Polymerase (Amersham Biosciences Corp., Piscataway,N.J.). Thermocycling was as follows: An initial denaturing step of 94°C. for 2 min (once), and 20 cycles of 94° C. for 30 sec (denature), 58°C. for 30 sec (anneal/extend); followed by a final extension step of 58°C. for 10 min (once). Again using the aforementioned Filtration Devices,the beads were washed briefly 3×400 μL with TE-50 mM NaCl then 3×400 μLMBG-Water. At this stage, beads could be stored by washing 1×400 μL in1×TE buffer containing 50% glycerol and 50 mM NaCl, then resuspending to5% beads (v/v) in the same buffer for storage at −20° C.

Next, the solid-phase bridge PCR product on the beads was hybridizedwith a fluorescently labeled complementary oligonucleotide correspondingto the SBE probe (i.e. primer). The SBE probe was commercially customsynthesized with a 5′ fluorescein label and PAGE purified by themanufacturer (Sigma-Genosys, The Woodlands, Tex.). The SBE probe wasdiluted to 5 μM final in TE-50 mM NaCl for hybridization experiments.Prior to use however, the 5 μM SBE probe solution was pre-clarified byspinning 1 min at maximum speed on a micro-centrifuge (˜13,000 rpm or˜16,000×g) and collecting the fluid supernatant. The supernatant wasthen passed though a Filtration Device (see earlier in this Example forFiltration Devices) and the filtrate saved for use as the SBE probesolution. The sequence of the SBE probe was as follows:

SBE Probe: 5′ [Fluorescein]gCACCCTAgAACCAAATCCAgCAgACTg3′ [SEQ NO. 46]

1 μL bead volume per sample was washed 2×400 μL with TE-50 mM NaCl usingthe aforementioned Filtration Devices. In the Filtration Device, each 1μL pellet corresponding to each sample was resuspended in 25 μL of theaforementioned clarified 5 μM SBE probe solution. The beads wereresuspended by manual pipetting then transferred to 0.5 mL polypropylenethin-wall PCR tubes. Hybridization was performed as follows in a PCRmachine: 2 min 94° C. (denature) (beads resuspended by vortex mixingjust before and at 2.5 min) followed by ramping down to 68° C. at a rateof 0.1° C./sec and subsequently holding 1 hour at 68° C. (anneal).

Just at the end of the above 1 hour 68° C. (anneal) step, while thetubes were still at 68° C. and still in the PCR machine, each sample wasrapidly diluted with 400 μL of 68° C. TE-50 mM NaCl, the suspensionsimmediately transferred to a Filtration Device and filtrationimmediately performed. The filtrate was then discarded. The beads werewashed 2×400 μL more with 68° C. TE-50 mM NaCl then 2×400 μL with roomtemperature TE-50 mM NaCl. Beads were lastly washed 2×400 μL with 50 mMNaCl. The beads were recovered from the Filtration Devices byresuspending the pellet in 50 μL of 50 mM NaCl and transferring to a 0.5mL polypropylene PCR tube. The beads were spun down in a standardmicro-centrifuge (just until reaches maximum speed of ˜13,000 rpmcorresponding to ˜16,000×g) and the fluid supernatant removed.

The 1 μL washed bead pellet was resuspended in 20 μL of 1×ThermoSequenase Reaction Buffer (150 mM Tris-HCl, pH 9.5, 67 mM MgCl₂)with 2.5 μM each of Cy3 labeled ddCTP and Cy5 labeled ddUTP, 25 μM eachof unlabeled ddATP and ddGTP, and 0.5 U/μL of the ThermoSequenase DNAPolymerase (Amersham Biosciences Corp., Piscataway, N.J.). The singlebase extension reaction was incubated for 20 min at 68° C. Again usingthe aforementioned Filtration Devices, the beads were washed briefly3×400 μL with 68° C. TE-50 mM NaCl then 1×400 μL with room temperatureTE-100 mM NaCl (10 mM Tris, pH 8.0, 1 mM EDTA, 100 mM NaCl).

Embedding the Beads in a Polyacrylamide Film and Fluorescence Imaging:

Lastly, the beads were embedded in a polyacrylamide film on a microscopeslide and fluorescently imaged to detect the bound Fluorescein labeledSBE probe as well as the Cy3 and Cy5 extension products corresponding tothe wild-type and mutant respectively. To do so, an Acrylamide Mix wasprepared by combining the following reagents in order: 244 μL of TE-100mM NaCl, 57 μL of 40% acrylamide (19:1 cross-linking) (Bio-RadLaboratories, Hercules, Calif.), 0.5 μL TEMED (Bio-Rad Laboratories,Hercules, Calif.), and 1 μL of a 10% (w/v) ammonium persulfate stock(prepared in MBG-Water from powder obtained from Bio-Rad Laboratories,Hercules, Calif.). Each aforementioned washed bead pellet was thenresuspended in 50 μL of the above Acrylamide Mix and combined by briefvortex mixing. 25 μL of the bead suspension was then pipetted to astandard glass microscope slide and overlaid with a standard 18 mmsquare microscope cover glass (coverslip). Polymerization was allowed tooccur for ˜10 min protected from light. Note that the adequately slowpolymerization process allows all beads to settle to the surface of themicroscope slide by unit gravity. When polymerization was complete,imaging was performed using an ArrayWoRX^(e) BioChip fluorescencemicroarray reader (Applied Precision, LLC, Issaquah, Wash.).

Results:

The fluorescence images are shown in FIG. 41 whereby the top pair ofimage panels correspond the minus template (blank) sample permutationand the bottom pair of image panels the plus template sample (50:50wild-type:mutant template mix initially added to beads at ratio of ˜1original template molecule initially added per each bead).

The top images in each panel pair (“SBE Probe Binding”) are a singlefluorescence channel corresponding to detection of binding of thefluorescein labeled SBE probe. Results show essentially no significantSBE probe binding in the case of the minus template blank. The presenceof beads in this sample however is confirmed by extremely weakauto-fluorescence (visible only at extremely high image intensitysettings; see inset box). Conversely, in the plus template sample,significant SBE probe binding is observed with an averagesignal-to-noise ratio of 33:1 (n>27 beads randomly sampled).

The bottom images of each panel pair (“SBE Probe Extension”) are 2-colorfluorescence image overlays corresponding to the wild-type (Cy3fluorescence channel; green) and mutant (Cy5 fluorescence channel; red)extension products. Compared to the minus template blank, the plustemplate beads have an average signal-to-noise ratio of 14:1 and 11:1for the Cy3 and Cy5 fluorescence channels respectively (n>50 beadssampled).

As a measure of relative mutant and wild-type APC content on each bead,the fluorescence images were quantified and the green:red fluorescenceratios calculated. Beads with a higher green:red ratio have a higherrelative wild-type content compared to beads with a lower green:redratio, and visa versa. Green:red ratios of a sampling of beads are shownin FIG. 41, indicated by arrows. Beads classified as wild-type had a 3-4fold higher green:red ratio than beads classified as mutant APC. Thenumber of beads classified as wild-type and mutant in the image shownapproximates the 50:50 ratio of wild-type and mutant template initiallyadded to the solid-phase bridge PCR reaction. Taken together, these datasuggest effective solid-phase bridge PCR amplification of one or a feworiginal template molecules per bead.

Example 45 Solid-Phase Bridge PCR for Multiplexed Detection ofMethylated DNA

DNA methylation, which silences genes via repression of transcriptionand also maintains genomic stability, occurs primarily on CpGdinucleotides at the C5 position of cytosine and plays a critical rolein both normal function of mammalian organisms as well as in disease(Reviewed in [Robertson. (2005) Nat Rev Genet. 6, 597-610]). Inparticular, aberrant DNA methylation has been associated with humancancers. Such methylation patterns aid in understanding the mechanismsof disease and act as specific biomarkers for molecularly baseddiagnostic or prognostic assays.

This Example pertains to the detection (analysis) of the methylationstatus of one or many regions of DNA, as biomarkers for colorectalcancer diagnostic or prognostic assays. The overall approach however, isnot intended to be limited to any one specific disease or specificbiomarker.

A multitude of analytical methods have been developed to detect DNAmethylation patterns in biological samples (e.g. reviewed in [Fraga &Esteller. (2002) Biotechniques 33, 632, 634, 636-649]). Despite thisvariety, virtually all methods are based on a few common principals:

First, current methods extract information on DNA methylation status byexploiting either methylation-sensitive/resistant/dependent restrictionenzymes (or nucleases) (e.g. [Singer-Sam et al. (1990) Nucleic Acids Res18, 687]) or sodium bisulfite conversion of DNA (e.g. [Frommuer et al.(1992) Proc Natl Acad Sci USA 89, 1827-1831]). The DNA cutting activityof methylation-sensitive restriction enzymes (or nucleases) is blockedby methylation, whereas methylation-resistant enzymes cut regardless ofmethylation state and methylation-dependent enzymes cut only if therecognition site(s) is methylated. On the other hand, sodium bisulfitetreatment converts unmethylated cytosines to uracils, whereas methylatedcytosines are protected from this chemical reaction, hence remaining ascytosines; thus creating methylation-dependent sequence differences.

Second, virtually all such methods subsequently utilize PCR either asthe detection step itself, or as a pre-amplification step prior todetection. Hence, these approaches are amenable to adaptation tosolid-phase bridge PCR based assays. The use of solid-phase bridge PCRaffords several advantages, including but not limited to: a) The abilityto multiplex, in a single solid-phase bridge PCR reaction, theamplification of various distinct biomarkers or b) the ability toperform amplification of 1 or a few template DNA molecules per eachbead, in order to facilitate for example, high sensitivity detection ofa few aberrantly methylated DNA molecules in the presence of an excessof normal.

In this Example, DNA from biological samples (e.g. stool, blood, plasma,serum, tissue or urine) of colorectal cancer patients (or normalpatients as controls) will be subjected tomethylation-sensitive/resistant/dependent restriction enzyme (ornuclease) digestion [e.g. using methylation-sensitive Hpa II, MspA1 I orHha I; or methylation-resistant Msp I; or methylation-dependent Dpn I orMcrBC enzymes from New England Biolabs, Inc., Ipswich, Mass.; ormethylation-dependent Gla I, Bls I, Bis I or Glu I enzymes fromSibEnzyme Ltd., Academtown, Russia]. Alternatively, sodium bisulfiteconversion of the sample DNA will be employed alone or will be used inconjunction with methylation-sensitive/resistant/dependent enzymedigestion.

In colorectal cancer, aberrant methylation patterns in the CDKN2A, MLH1,HTLF, SLC5A8, RASSF2A and vimentin genes, among others, have beenidentified and have diagnostic potential [Kane et al. (1997) Cancer Res57, 808-811; Ahuja et al. (1997) Cancer Res 57, 3370-3374; Moinova etal. (2002) Proc Natl Acad Sci USA 99, 4562-4567; Li et al. (2003) ProcNatl Acad Sci USA 100, 8412-8417; Chen et al. (2005) J Natl Cancer Inst97, 1124-1132; Hesson et al. (2005) Oncogene 24, 3987-3994]. Such genes,and their relevant regions of aberrant methylation will be targeted inthis Example, based on the literature reports. In some cases, multiplegenes (biomarkers) or segments thereof, will be targeted in a singlesolid-phase bridge PCR reaction, to facilitate multiplexing henceincreasing specificity and sensitivity of the colorectal cancerdiagnostic or prognostic assays.

In one scenario, a methylation-sensitive enzyme will be selected thatdoes not digest the targeted template DNA region if methylated, allowingsubsequent amplification of the targeted methylated region bysolid-phase bridge PCR. Separately, a methylation-resistant ormethylation-dependent enzyme will also be selected that cuts within thetemplate DNA region targeted by the solid-phase bridge PCR primers,either regardless of methylation status or only if methylation ispresent, thereby preventing amplification of the targeted methylatedregion by solid-phase bridge PCR. Positive formation of solid-phasebridge PCR product in the former case, coupled with lack of (or reduced)formation of solid-phase bridge PCR product in the latter case indicatesthe targeted region is methylated. Sample DNA not treated with anyrestriction enzymes (or nucleases), as well as fully methylated orunmethylated DNA treated with restriction enzymes (or nucleases) will beused as additional controls. Alternatively, following digestion with anysuch enzymes, the cut ends of the DNA (one or both ends) will beselectively attached to oligonucleotide adaptors to facilitatedownstream amplification of the targeted region by directing at leastone of the solid-phase bridge PCR primers against at least one adaptor(other primer will be biomarker region specific). Again, depending onthe enzymes selected, formation or lack of (or reduced) formation ofsolid-phase bridge PCR product will indicate the methylation status.Enzyme treatments will be performed similar to as described in thescientific literature (e.g. [Singer-Sam et al. (1990) Nucleic Acids Res18, 687; Liu et al. (2002) Otolaryngol Head Neck Surg 126, 548-553;Badal et al. (2003) J Virol 77, 6227-6234]). Solid-phase bridge PCR willbe performed (individually or multiplexed for various biomarkers in onereaction) either as in Example 31 (where initial soluble template DNA ispresent throughout the entire solid-phase bridge PCR amplification) orExamples 36-41 (where after initial capture of the soluble template DNAonto the primer-coated beads and extension of the primers once, theinitial template DNA is washed away prior to subsequent solid-phasebridge PCR amplification). Detection of the solid-phase bridge PCRproduct (amplicon) will be directly on the beads at the DNA level, vialabeling with fluorescence deoxynucleotides during the solid-phasebridge PCR reaction (e.g. as in Example 36) or probing withfluorescently labeled complementary oligonucleotides (e.g. as in Example38). Alternatively, detection will be indirect, by cell-free expressionof the solid-phase bridge PCR product into protein, capture of theprotein onto the same beads, in some cases contact-photo transfer of theproteins onto a second surface and detection of the protein, either onor off the original beads. Detection in this case will be via antibody(e.g. readout by microarray reader as in Example 40 or by flow cytometryas in Example 41) or mass spectrometry (e.g. Example 34).

In a second scenario, enzyme digestion will not be performed, instead,the sample DNA will be subjected to sodium bisulfite conversion tocreate methylation-dependent sequence differences. Sodium bisulfitetreatment will be performed according to the scientific literature (e.g.[Frommer et al. (1992) Proc Natl Acad Sci USA 89, 1827-1831]). Fullyunmethylated or fully methylated DNA, as well as DNA not treated withsodium bisulfite will be used as additional controls. Solid-phase bridgePCR will then be performed on all samples (individually or multiplexedfor various biomarkers in one reaction) using primers that specificallytarget the methylation-dependent sequence differences (i.e. afterbisulfite treatment, primers target unconverted and therefore methylatedsequences) (called Methylation-Specific PCR or MSP). In this case,positive formation of solid-phase bridge PCR product indicates thetargeted region(s) is methylated. Detection of the solid-phase bridgePCR product will be directly at the DNA level or following expression ofthe DNA into protein, as detailed above for themethylation-sensitive/resistant/dependent enzyme digestion approach.Alternatively, the bisulfite treated DNA will be amplified viasolid-phase bridge PCR using primers which do not target any potentiallymethylated regions, and detection of the methylation-dependent sequencedifferences will be achieved by various methods including restrictionenzyme digestion (COBRA), single nucleotide primer extension (Ms-SNuPE)or DNA sequencing according to the scientific literature [Frommer et al.(1992) Proc Natl Acad Sci USA 89, 1827-1831; Gonzalgo & Jones. (1997)Nucleic Acids Res 25, 2529-2531; Xiong & Laird. (1997) Nucleic Acids Res25, 2532-2534].

Example 46 Solid-Phase Bridge PCR for Multiplexed Detection ofMethylated DNA in Vimentin and RASSF2A Markers for Colorectal CancerDiagnosis

Methylation of the vimentin and RASSF2A markers and the detection ofcolorectal cancer, using methylation-specific PCR (MSP), is reported inthe scientific literature [Chen et al. (2005) J Natl Cancer Inst 97,1124-1132; Hesson et al. (2005) Oncogene 24, 3987-3994; Park et al.(2007) Int J Cancer 120, 7-12]. This Example will demonstrate theability to use solid-phase bridge PCR to multiplex MSP assays formultiple diagnostic markers, such as the vimentin and RASSF2A markersshown here.

Preparation of Agarose Beads Covalently Conjugated to PCR Primers Usedfor Solid-Phase Bridge PCR:

Production of Primer-Conjugated Agarose Beads will be performed as inExample 36 except beads with the following primer pairs will be prepared(each primer pair bead set prepared separately).

Vimentin Unmethylated Primer Pair: [SEQ NO. 47] Forward:5′[Amine]TTgAggTTTTTgTgTTAgAgATgTAgTTgT3′ [SEQ NO. 48] Reverse:5′[Amine]ACTCCAACTAAAACTCAACCAACTCACA3′ Vimentin Methylated Primer Pair:[SEQ NO. 49] Forward: 5′[Amine]TCgTTTCgAggTTTTCgCgTTAgAgAC3′ [SEQ NO.50] Reverse: 5′[Amine]CgACTAAAACTCgACCgACTCgCgA3′ RASSF2A UnmethylatedPrimer Pair: [SEQ NO. 51] Forward: 5′[Amine]AgTTTgTTgTTgTTTTTTAggTgg3′[SEQ NO. 52] Reverse: 5′[Amine]AAAAAACCAACAACCCCCACA3′ RASSF2AMethylated Primer Pair: [SEQ NO. 53] Forward:5′[Amine]AgTTCgTCgTCgTTTTTTAggC3′ [SEQ NO. 54] Reverse:5′[Amine]AAAAACCAACgACCCCCgCg3′

Qualitative Analysis of Primer Attachment:

Performed as in Example 36

Template for Solid-Phase Bridge PCR and Bisulfite Conversion:

Fully methylated genomic DNA (CpGenome™ Universal Methylated DNA;Chemicon-Millipore, Billerica, Mass.), i.e. “mutant” DNA, and normalhuman blood genomic DNA (wild-type DNA; i.e. unmethylated at vimentinand RASSF2A marker regions) (Clontech, Mountain View, Calif.) will bepurchased commercially to be used as the template solid-phase bridgePCR.

Prior to bisulfite conversion, genomic DNA will be mechanicallyfragmented into an average size of roughly 500 bp via direct probesonication. Fragmentation will be verified by standard agarose gelelectrophoresis. The fragmented genomic DNA will then be mixed in thefollowing ratios of wild-type (unmethylated) to “mutant” (methylated):0:100, 50:50, 95:5, 99:1, 99.9:0.1, 99.99:0.01 and 100:0.

For bisulfite conversion, the aforementioned fragmented DNA mixtureswill first be denatured by preparing the following reaction: 12.5 ng/μLsingle-stranded carrier DNA (lambda DNA, E. coli genomic DNA or salmonsperm DNA), 0.3N NaOH, and 1-50 ng of the aforementioned fragmentedgenomic DNA mixtures. The denaturation reaction will then be incubatedfor 10 min at 37° C. Next, 30 μL of 10 mM hydroquinone will be added (10mM hydroquinone prepared fresh from 25× stock which is stored at −20°C.) followed by 500 μL of a 3M sodium bisulfite stock (stock adjusted topH 5.0 with NaOH). Lastly, 200 μL of mineral oil will be added and thereaction will be incubated at 50° C. for 16 hrs.

The resultant bisulfite converted DNA will be purified using thecommercially available Wizard® DNA Clean-Up System (Promega, Madison,Wis.) according to the manufacturer's instructions. After elution fromthe Wizard® DNA Clean-Up System mini-columns in 90 μL 0.1×TE (1 mMTris-HCl, pH 8.0, 0.1 mM EDTA), the DNA will be ethanol precipitated.Ethanol precipitation will be carried out as follows: 45 μL of 1 N NaOHwill be added to each sample and briefly vortex mixed. After 5 min, 15μL of 3M sodium acetate, pH 5.2, will be added to each tube. Next, 1 μLof 20 mg/mL glycogen will be added followed by 300 μL of ethanol. Themixture will then be incubated at −80° C. for 20 min and spun in amicro-centrifuge for 10 min (maximum speed of ˜13,000 rpm correspondingto ˜16,000×g). The ethanol will be removed and the DNA pellet air driedat room temperature for 15 min. The DNA will then be re-dissolved in0.1×TE for immediate use or storage at −20° C.

Solid-Phase Bridge PCR:

2.5 μL actual total bead volume of the previously preparedPrimer-Conjugated Agarose Beads will be used per each sample. The 2.5 μLbeads for each sample will be comprised of equal quantities of each ofthe 4 aforementioned Primer-Conjugated Agarose Bead species (0.625 μLeach of vimentin and RASSF2A primer pair beads, methylated andunmethylated directed versions) to allow multiplexed solid-phase bridgePCR of the 2 markers. First, enough beads for all sample permutationswill be pre-washed in bulk. Beads will be washed using 0.45 micron poresize, PVDF membrane, micro-centrifuge Filtration Devices (Ultrafree-MCDurapore Micro-centrifuge Filtration Devices, 400 μL capacity;Millipore, Billerica, Mass.). Unless otherwise noted, all washesinvolving the Filtration Devices will be by brief vortex mixing (˜5sec), spinning down briefly in a micro-centrifuge (just until reachesmaximum speed of ˜13,000 rpm corresponding to ˜16,000×g) and discardingof the filtrate. Beads will first be washed 2×400 μL with TE-50 mM NaCl(10 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl). Beads will then beresuspended in TE-50 mM NaCl to 20% (v/v) and the suspension recoveredinto a 0.5 mL thin-walled polypropylene PCR tube. The tube will beplaced in a PCR machine at 95° C. for 10 min to allow heat-mediatedwashing (lid temperature 105° C. and no mineral oil used) (beads wereresuspended by brief gentle vortex mixing just before this step). Afterheating, the tube will immediately be removed from the PCR machine, thebeads will be diluted to 400 μL with TE-50 mM NaCl and the beadsuspension will then be transferred to a Filtration Device. Filtrationwill be performed and the filtrate will be discarded. Beads will bebriefly washed 1×400 μL more with TE-50 mM NaCl then 1×400 μL withMBG-Water.

Following the final filtration step on the bead samples, the bulk washedbead pellet will be resuspended in a commercially available PCR reactionmixture (HotStarTaq DNA Polymerase; Qiagen, Valencia, Calif.) which willprepared according to the manufacturer's instructions except with a 0.2U/μL final DNA polymerase concentration and no soluble primers (and notemplate added yet). Additionally, a fluorescence BODIPY-FL-dUTP reagentwill also be added to a 20 μM final concentration from themanufacturer's 1 mM stock (ChromaTide® BODIPY® FL-14-dUTP; InvitrogenCorporation, Carlsbad, Calif.), in order to achieve subsequentfluorescence labeling of the PCR amplicon (PCR product). The beads willbe resuspended with 10 μL of PCR reaction mixture per each 1 μL actualbead volume. The suspension will then be recovered from the FiltrationDevice into fresh 0.5 mL polypropylene thin-wall PCR tubes, divided upat 25 μL total suspension volume per tube (i.e. per sample). At thispoint, 1-2 μL of the various fragmented genomic DNA template mixtureswill be added to the appropriate tubes (a minus template negativecontrol will also be performed). The samples will be subjected to thefollowing thermocycling in a PCR machine (lid temperature 105° C. and nomineral oil used): An initial denaturing step (once) of 95° C. for 15min (beads will briefly be resuspended by gentle vortex mixing justbefore this step), and 40 cycles of 94° C. for 30 sec (denature), 58° C.for 30 sec (anneal) and 72° C. for 30 sec (extend); followed by a finalextension step of 72° C. for 5 min.

400 μL of TE-50 mM NaCl-T (10 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl,0.01% v/v Tween-20) will be added to each completed solid-phase bridgePCR reaction and the suspensions transferred to fresh 0.5 mLpolypropylene PCR tubes. The beads will then be spun down in amicro-centrifuge (just until reaches maximum speed of ˜13,000 rpmcorresponding to ˜16,000×g) and the fluid supernatant carefully removed.The beads will be washed 3×400 μL more with TE-50 mM NaCl-T;resuspending by ˜5 sec vortex mixing then spinning down and discardingthe fluid supernatant as above. Following the final wash, as much of thefluid supernatant as possible will be removed from the bead pellet bymanual pipetting. The beads will be lastly resuspended to 5% (v/v) usingSP-PCR Storage Buffer (10 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl, all in50% v/v glycerol).

If necessary, a second full round of solid-phase bridge PCR will beperformed to increase product formation to detectible levels. To do so,the bead samples will be washed 2×400 μL with MBG-Water using aFiltration Device as described earlier in this Example. Following thefinal filtration step on the bead samples, each washed bead pellet willbe resuspended in 25 μL of a fresh batch of PCR reaction mixture asdetailed above in this Example (again containing the BODIPY-FL-dUTPreagent). The suspensions will then be recovered from their FiltrationDevices into fresh 0.5 mL polypropylene thin-wall PCR tubes and againsubjected to thermocycling as detailed above in this Example (40cycles). After the second round of solid-phase bridge PCR thermocyclingis complete, the beads will again be washed. To do so, 400 μL of TE-50mM NaCl-T (10 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl, 0.01% v/vTween-20) will be added to each completed solid-phase bridge PCRreaction and the suspensions transferred to fresh 0.5 mL polypropylenePCR tubes. The beads will then be spun down in a micro-centrifuge (justuntil reaches maximum speed of ˜13,000 rpm corresponding to ˜16,000×g)and the fluid supernatant carefully removed. The beads will be washed3×400 μL more with TE-50 mM NaCl-T; resuspending by ˜5 sec vortex mixingthen spinning down and discarding the fluid supernatant as above.Following the final wash, as much of the fluid supernatant as possiblewill be removed from the bead pellet by manual pipetting. Beads are thenresuspended to 5% (v/v) in SP-PCR Storage Buffer (10 mM Tris, pH 8.0, 1mM EDTA, 50 mM NaCl, all in 50% v/v glycerol) for storage at −20° C.

Oligonucleotide Hybridization Probing:

Fluorescently labeled oligonucleotide probes will be commercially customsynthesized and HPLC purified by the manufacturer (Sigma-Genosys, TheWoodlands, Tex.). The probes will be reconstituted to 100 μM inMBG-Water and further desalted using MicroSpin G-25 columns according tothe manufacturer's instructions (Amersham Biosciences Corp., Piscataway,N.J.), except that the columns will be pre-washed 2×350 μL withMBG-Water prior to sample loading (to wash, columns will be mixedbriefly in the MBG-Water then spun 1 min in a standard micro-centrifugeat the proper speed). The probes will be diluted to 5 μM final in TE-50mM NaCl for hybridization experiments. Prior to use however, the 5 μMprobe solution will be pre-clarified by spinning 1 min at maximum speedon a micro-centrifuge (˜13,000 rpm or ˜16,000×g) and collecting thefluid supernatant. The supernatant will then be passed though aFiltration Device (see earlier in this Example for Filtration Devices)and the filtrate will be saved for use as the probe solution.

In this Example, simultaneous dual probing will be performed by creatinga single probing solution containing 5 μM of each probe, labeled ontheir 5′ ends with the Cy3 or Cy5 fluorophores by the manufacturer(Sigma-Genosys, The Woodlands, Tex.). The gene-specific probes willcomplementary to an internal segment of the vimentin and RASSF2Asolid-phase bridge PCR amplicons, corresponding to the “mutant” (i.e.methylated) bisulfite converted forms:

Human Vimentin Methylated & Bisulfite Converted:5′[Cy3]gTAggATgTTCggCggTTCg3′ [SEQ NO. 55] Human RASSF2A Methylated &Bisulfite Converted: 5′[Cy5]gTTTTAgTTTTCggCgCggg3′ [SEQ NO. 56]

Following completion of all prior solid-phase bridge PCR reaction stepsin this Example, 20 μL of the aforementioned stored 5% (v/v) stock beadsuspension (i.e. 1 μL post-PCR stored beads) will be taken and washed2×400 μL with TE-50 mM NaCl using the aforementioned Filtration Devices.In the Filtration Device, each 1 μL pellet corresponding to each samplewill be resuspended in 25 μL of the aforementioned clarified 5 μM probesolution. The beads will be resuspended by manual pipetting thentransferred to 0.5 mL polypropylene thin-wall PCR tubes. Hybridizationwill be performed as follows in a PCR machine (lid temperature always105° C., no mineral oil used): 5 min 95° C. (denature) (beads will beresuspended by vortex mixing just before this step) followed by rampingdown to 60° C. at a rate of 0.1° C./sec and subsequently holding 1 hourat 60° C. (anneal).

Just at the end of the above 1 hour 60° C. (anneal) step, while thetubes are still at 60° C. and still in the PCR machine, each sample willbe rapidly diluted with 400 μL of 60° C. TE-50 mM NaCl, the suspensionsimmediately transferred to a Filtration Device and filtrationimmediately performed. The filtrate will then be discarded. The beadswill be washed 3×400 μL more with room temperature TE-50 mM NaCl then1×400 μL with room temperature TE-100 mM NaCl (10 mM Tris, pH 8.0, 1 mMEDTA, 100 mM NaCl). The beads will be recovered from the FiltrationDevices by resuspending the pellets in 50 μL of TE-100 mM NaCl andtransferring to a 0.5 mL polypropylene PCR tube. The beads will be spundown in a standard micro-centrifuge (just until reaches maximum speed of˜13,000 rpm corresponding to ˜16,000×g) and the fluid supernatant willbe removed.

Embedding the Beads in a Polyacrylamide Film and Fluorescence Imaging:

Lastly, the beads will be embedded in a polyacrylamide film on amicroscope slide and fluorescently imaged to detect the bound Cy3 andCy5 labeled hybridization probes. To do so, an Acrylamide Mix will beprepared by combining the following reagents in order: 244 μL of TE-100mM NaCl, 57 μL of 40% acrylamide (19:1 cross-linking) (Bio-RadLaboratories, Hercules, Calif.), 0.5 μL TEMED (Bio-Rad Laboratories,Hercules, Calif.), and 1 μL of a 10% (w/v) ammonium persulfate stock(prepared in MBG-Water from powder obtained from Bio-Rad Laboratories,Hercules, Calif.). Each aforementioned washed bead pellet will then beresuspended in 50 μL of the above Acrylamide Mix and combined by briefvortex mixing. 25 μL of the bead suspension will then be pipetted to astandard glass microscope slide and overlaid with a standard 18 mmsquare microscope cover glass (coverslip). Polymerization will beallowed to occur for ˜10 min protected from light. Note that theadequately slow polymerization process will allow all beads to settle tothe surface of the microscope slide by unit gravity. When polymerizationis complete, imaging will be performed using an ArrayWoRx^(e) BioChipfluorescence microarray reader (Applied Precision, LLC, Issaquah,Wash.). The beads will be imaged in 3 different fluorescence channels todetect the BODIPY-FL dUTP labels as well as the Cy3 and Cy5hybridization probes.

Results:

Results are anticipated to show proof-of-principal for multiplexing MSPof several disease biomarkers, in this case for colorectal cancer, byusing a single solid-phase bridge PCR reaction. Multiplexing is achievedby using a mixture of PCR primer coated beads in the single solid-phasebridge PCR reaction, with each bead species targeting the variousbiomarkers, methylated or unmethylated versions (following bisulfiteconversion) (i.e. 4 bead species in this case, with a multitudereplicates of each bead species). In this Example, “mutant” (methylated;bisulfite converted) vimentin and RASSF2A amplicons are detected ontheir corresponding beads using selective complementary hybridizationprobes, each labeled with a different fluorophore (Cy3 and Cy5). Theseamplicons will also carry the BODIPY-FL dUTP fluorescence label.Selective formation of PCR product on these beads indicates the aberrantmethylated state of at least a fraction of the biomarker present in thesample and hence the presence of disease (in the case where actualpatient samples are assayed). Wild-type (unmethylated; bisulfiteconverted) vimentin and RASSF2A amplicons are detected on theircorresponding beads by the presence of the BODIPY-FL dUTP fluorescencelabel, but absence of any hybridization probe signal. Detection of thewild-type amplicons serves only as a positive control and could bedetected more specifically using additional hybridization probes bearingdifferent fluorophores (in which case BODIPY-FL dUTP fluorescencelabeling could be omitted). Because the methylation directed primerbeads will selectively amplify any methylated biomarker present in thesample, detection of very low percentages of the methylated (“mutant”)biomarker is expected among a large background of wild-type(unmethylated). In this Example, detection is expected at ratios atleast as low as 1 “mutant” (methylated) DNA biomarker molecule out ofevery 10,000 DNA biomarker molecules (9,999 wild-type DNA biomarkermolecules).

Note that the method in this Example could be modified to provide foreffective solid-phase bridge PCR amplification of single DNA moleculesper bead, in which case the initially added soluble template DNA iswashed out following a single primer extension step on the solid-phasebridge PCR beads (e.g. as done in Examples 36-39 for instance). Thiswould be expected to reduce background from unintended amplification ofwild-type DNA on “mutant” (methylated) directed primer beads (vianon-specific hybridization).

Ultimately, in the case where a multitude of biomarkers are to bemultiplexed (e.g. more than 2), the beads themselves could carry aunique readable intrinsic code which identifies the specific primer pairon each bead. Different hybridization probes, specifically directedagainst the different biomarker amplicons, could be used tosimultaneously probe the bead population. In this case, the differenthybridization probes could all carry the same fluorophore (or otherreporter), while determination of which biomarkers were positivelyamplified could be made by reading the code of the specific primercoated bead species. Coded bead platforms manufactured by LuminexCorporation (Austin, Tex.) and Illumina Incorporated (San Diego,Calif.), for example, would be suitable for this purpose.

Example 47 Solid-Phase Bridge PCR for Multiplexed Detection of BisulfiteConverted Wild-Type Vimentin and RASSF2A DNA Markers: Applications inColorectal Cancer Diagnosis

Methylation of the vimentin and RASSF2A markers and the detection ofcolorectal cancer, using methylation-specific PCR (MSP), is reported inthe scientific literature [Chen et al. (2005) J Natl Cancer Inst 97,1124-1132; Hesson et al. (2005) Oncogene 24, 3987-3994; Park et al.(2007) Int J Cancer 120, 7-12]. This Example demonstrates the ability touse solid-phase bridge PCR to multiplex MSP assays for multiplediagnostic markers. In this Example, multiplexed detection of thewild-type vimentin and RASSF2A markers is demonstrated. However, thetechnique is equally applicable to the detection of the “mutant”, i.e.methylated markers, simply by changing the primer sequences.

Preparation of Agarose Beads Covalently Conjugated to PCR Primers Usedfor Solid-Phase Bridge PCR:

Production of Primer-Conjugated Agarose Beads was performed as inExample 36 except beads with the following primer pairs were prepared(each primer pair bead set prepared separately).

Vimentin Unmethylated Primer Pair: [SEQ NO. 57] Forward:5′[Amine]TTgAggTTTTTgTgTTAgAgATgTAgTTgT3′ [SEQ NO. 58] Reverse:5′[Amine]ACTCCAACTAAAACTCAACCAACTCACA3′ RASSF2A Unmethylated PrimerPair: [SEQ NO. 59] Forward: 5′[Amine]AgTTTgTTgTTgTTTTTTAggTgg3′ [SEQ NO.60] Reverse: 5′[Amine]AAAAAACCAACAACCCCCACA3′

Qualitative Analysis of Primer Attachment:

Performed as in Example 36

Template for Solid-Phase Bridge PCR and Bisulfite Conversion:

Normal human blood genomic DNA (wild-type DNA; i.e. umethylated atvimentin and RASSF2A marker regions) (Clontech, Mountain View, Calif.)was purchased commercially. For bisulfite conversion, the normal humanblood genomic DNA was first denatured by preparing the followingreaction: 12.5 ng/μL single-stranded carrier DNA (lambda DNA, E. coligenomic DNA or salmon sperm DNA), 0.3N NaOH, and 1-50 ng of theaforementioned normal human blood genomic DNA. The denaturation reactionwas then incubated for 10 min at 37° C. Next, 30 μL of 10 mMhydroquinone was added (10 mM hydroquinone prepared fresh from 25× stockwhich is stored at −20° C.) followed by 500 μL of a 3M sodium bisulfitestock (stock adjusted to pH 5.0 with NaOH). Lastly, 200 μL of mineraloil was added and the reaction incubated at 50° C. for 16 hrs.

The resultant bisulfite converted DNA was purified using thecommercially available Wizard® DNA Clean-Up System (Promega, Madison,Wis.) according to the manufacturer's instructions. After elution fromthe Wizard® DNA Clean-Up System mini-columns in 90 μL 0.1×TE (1 mMTris-HCl, pH 8.0, 0.1 mM EDTA), the DNA was ethanol precipitated.Ethanol precipitation was carried out as follows: 45 μL of 1 N NaOH wasadded to each sample and briefly vortex mixed. After 5 min, 15 μL of 3Msodium acetate, pH 5.2, was added to each tube. Next, 1 μL of 20 mg/mLglycogen was added followed by 300 μL of ethanol. The mixture was thenincubated at −80° C. for 20 min and spun in a micro-centrifuge for 10min (maximum speed of ˜13,000 rpm corresponding to ˜16,000×g). Theethanol was removed and the DNA pellet air dried at room temperature for15 min. The DNA was then re-dissolved in 0.1×TE for immediate use orstorage at −20° C.

Following bisulfite conversion of the normal human blood genomic DNA asdescribed above in this Example, standard solution-phase PCR wasperformed with the following MSP primers directed against the bisulfiteconverted wild-type DNA markers:

Vimentin Unmethylated Primer Pair: [SEQ NO. 61] Forward:5′TTgAggTTTTTgTgTTAgAgATgTAgTTgT3′ [SEQ NO. 62] Reverse:5′ACTCCAACTAAAACTCAACCAACTCACA3′ RASSF2A Unmethylated Primer Pair: [SEQNO. 63] Forward: 5′AgTTTgTTgTTgTTTTTTAggTgg3′ [SEQ NO. 64] Reverse:5′AAAAAACCAACAACCCCCACA3′

The standard solution-phase PCR was carried out in a commerciallyavailable PCR reaction mixture (HotStarTaq DNA Polymerase; Qiagen,Valencia, Calif.) which was prepared according to the manufacturer'sinstructions. The reactions were subjected to the followingthermocycling in a PCR machine: An initial denaturing step (once) of 95°C. for 15 min, and 40 cycles of 94° C. for 30 sec (denature), 58° C. for30 sec (anneal) and 72° C. for 30 sec (extend); followed by a finalextension step of 72° C. for 5 min. The PCR products from the MSPreaction were purified by agarose gel electrophoresis using standardpractices and these purified products were used as template forsolid-phase bridge PCR as described below.

Solid-Phase Bridge PCR:

2.5 μL actual total bead volume of the previously preparedPrimer-Conjugated Agarose Beads was used per each sample. The 2.5 μLbeads for each sample was comprised of equal quantities of each of the 2aforementioned Primer-Conjugated Agarose Bead species (1.25 μL each ofvimentin and RASSF2A primer pair beads, umethylated directed versions)to allow multiplexed solid-phase bridge PCR of the 2 markers. Additionalnon-multiplexed sample permutations were prepared which comprised 2.5 μLbead volume of either the vimentin or the RASSF2A primer pair beadsonly. Beads were pre-washed to remove any non-covalently attachedprimers. Beads were initially washed using 0.45 micron pore size, PVDFmembrane, micro-centrifuge Filtration Devices (Ultrafree-MC DuraporeMicro-centrifuge Filtration Devices, 400 μL capacity; Millipore,Billerica, Mass.). Unless otherwise noted, all washes involving theFiltration Devices were by brief vortex mixing (˜5 sec), spinning downbriefly in a micro-centrifuge (just until reaches maximum speed of˜13,000 rpm corresponding to ˜16,000×g) and discarding the filtrate.Initial washes were 2×400 μL with TE-50 mM NaCl (10 mM Tris, pH 8.0, 1mM EDTA, 50 mM NaCl). Beads were then resuspended in TE-50 mM NaCl to20% (v/v) and the suspensions recovered into 0.5 mL thin-walledpolypropylene PCR tubes. The tubes were placed in a PCR machine at 95°C. for 10 min to allow heat-mediated washing (lid temperature 105° C.and no mineral oil used) (beads were resuspended by brief gentle vortexmixing just before this step). After heating, the tubes were immediatelyremoved from the PCR machine, the beads were diluted to 400 μL withTE-50 mM NaCl and the bead suspensions were then transferred toFiltration Devices. Filtration was performed and the filtrate discarded.Beads were briefly washed 1×400 μL more with TE-50 mM NaCl then 1×400 μLwith MBG-Water.

Following the final filtration step (wash) on the bead samples, thewashed bead pellets were resuspended in a commercially available PCRreaction mixture (HotStarTaq DNA Polymerase; Qiagen, Valencia, Calif.)which was prepared according to the manufacturer's instructions exceptwith a 3 mM total magnesium concentration and no soluble primers (and notemplate added yet). The beads were resuspended with 10 μL of PCRreaction mixture per each 1 μL actual bead volume. The suspensions (˜25μL) were placed into 0.5 mL polypropylene thin-wall PCR tubes. At thispoint, 1 μL of the aforementioned template DNA was added (a minustemplate negative control was also performed). The resultant templateconcentration was 0.4 ng/μL and was a 50:50 mix of the vimentin andRASSF2A templates for the multiplexed sample (0.2 ng/μL final of eachtemplate for 0.4 ng/μL total template). 0.4 ng/μL of the correspondingsingle template species was used for the non-multiplexed samples. Forthe multiplexed sample, this resulted in a ratio of 28,000 and 54,000attomoles of template per μL of actual Primer-Conjugated Agarose Beadvolume for vimentin and RASSF2A respectively. With 1 μL ofPrimer-Conjugated Agarose Beads determined to contain approximately1,000 beads, 28,000 and 54,000 attomoles of template per μL of beadsrepresents a ratio of approximately 2×10⁷ and 3×10⁷ template moleculesadded per bead for vimentin and RASSF2A respectively, in the multiplexedsample (beads physically enumerated under a microscope both in diluteddroplets of bead suspension and with suspensions in a hemacytometer cellcounting chamber). The samples were subjected to the followingthermocycling in a PCR machine (lid temperature 105° C. and no mineraloil used): An initial denaturing step (once) of 95° C. for 15 min (beadswere briefly resuspended by gentle vortex mixing just before this step),and 40 cycles of 94° C. for 30 sec (denature), 58° C. for 2 min (anneal)and 72° C. for 1 min (extend); followed by a final extension step of 72°C. for 5 min.

400 μL of TE-50 mM NaCl (10 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl) wasadded to each completed solid-phase bridge PCR reaction and thesuspensions transferred to Filtration Devices (Ultrafree-MC DuraporeMicro-centrifuge Filtration Devices, 400 μL capacity; Millipore,Billerica, Mass.). Filtration was performed in a micro-centrifuge (justuntil reaches maximum speed of ˜13,000 rpm corresponding to ˜16,000×g)and the filtrate discarded. The beads were washed 3×400 μL more withTE-50 mM NaCl; resuspending by ˜5 sec vortex mixing then performingfiltration and discarding the filtrate as described earlier in thisExample.

Oligonucleotide Hybridization Probing:

Fluorescently labeled oligonucleotide probes were commercially customsynthesized and HPLC purified by the manufacturer (Sigma-Genosys, TheWoodlands, Tex.). The probes were reconstituted to 100 μM in MBG-Waterand further desalted using MicroSpin G-25 columns according to themanufacturer's instructions (Amersham Biosciences Corp., Piscataway,N.J.), except that the columns were pre-washed 2×350 μL with MBG-Waterprior to sample loading (to wash, columns were mixed briefly in theMBG-Water then spun 1 min in a standard micro-centrifuge at the properspeed). The probes were diluted in TE-50 mM NaCl for hybridizationexperiments. Prior to use however, the diluted probe solution waspre-clarified by spinning 1 min at maximum speed on a micro-centrifuge(˜13,000 rpm or ˜16,000×g) and collecting the fluid supernatant. Thesupernatant was then passed though a Filtration Device (see earlier inthis Example for Filtration Devices) and the filtrate was saved for useas the clarified probe solution.

In this Example, simultaneous dual hybridization probing was performedby creating a single probing solution containing 1 μM of each probe,labeled on their 5′ ends with the Cy3 or Cy5 fluorophores by themanufacturer (Sigma-Genosys, The Woodlands, Tex.). The gene-specificprobes were complementary to an internal segment of the vimentin andRASSF2A solid-phase bridge PCR amplicons:

Human Vimentin Unmethylated & Bisulfite Converted:5′[Cy3]TgTAggATgTTTggTggTTTggg3′ [SEQ NO. 65] Human RASSF2A Unmethylated& Bisulfite Converted: 5′[Cy5]TTTTggTgTggggAggTggT3′ [SEQ NO. 66]After solid-phase bridge PCR and washing of the beads as describedearlier in this Example, the bead pellets corresponding to each samplewere resuspended in 25 μL of the aforementioned clarified probe solution(containing both probes, for vimentin and RASSF2A). The beads wereresuspended by manual pipetting then transferred to 0.5 mL polypropylenethin-wall PCR tubes. Hybridization was performed as follows in a PCRmachine (lid temperature always 105° C., no mineral oil used): 5 min 95°C. (denature) (beads were be resuspended by vortex mixing just beforethis step) followed by ramping down to 60° C. at a rate of 0.1° C./secand subsequently holding 1 hour at 60° C. (anneal).

Just at the end of the above 1 hour 60° C. (anneal) step, while thetubes were still at 60° C. and still in the PCR machine, each sample wasrapidly diluted with 400 μL of 60° C. TE-50 mM NaCl, the suspensionsimmediately transferred to a Filtration Device and filtrationimmediately performed. The filtrate was then discarded. The beads werewashed 2×400 μL more with 60° C. TE-50 mM NaCl then 1×400 μL with roomtemperature TE-50 mM NaCl. Beads were lastly washed 1×400 μL with TE-100mM NaCl (10 mM Tris, pH 8.0, 1 mM EDTA, 100 mM NaCl). The beads wererecovered from the Filtration Devices by resuspending the pellets in 50μL of TE-100 mM NaCl and transferring to a 0.5 mL polypropylene PCRtube. The beads were spun down in a standard micro-centrifuge (justuntil reaches maximum speed of ˜13,000 rpm corresponding to ˜16,000×g)and the fluid supernatant was removed.

Embedding the Beads in a Polyacrylamide Film and Fluorescence Imaging:

Lastly, the beads were embedded in a polyacrylamide film on a microscopeslide and fluorescently imaged to detect the bound Cy3 and Cy5 labeledhybridization probes. To do so, an Acrylamide Mix was prepared bycombining the following reagents in order: 244 μL of TE-100 mM NaCl, 57μL of 40% acrylamide (19:1 cross-linking) (Bio-Rad Laboratories,Hercules, Calif.), 0.5 μL TEMED (Bio-Rad Laboratories, Hercules,Calif.), and 1 μL of a 10% (w/v) ammonium persulfate stock (prepared inMBG-Water from powder obtained from Bio-Rad Laboratories, Hercules,Calif.). Each aforementioned washed bead pellet was then resuspended to2% (v/v) beads in the above Acrylamide Mix and combined by brief vortexmixing. 25 μL of the bead suspension was then pipetted to a standardglass microscope slide and overlaid with a standard 18 mm squaremicroscope cover glass (coverslip). Polymerization was allowed to occurfor ˜10 min protected from light. Note that the adequately slowpolymerization process allows all beads to settle to the surface of themicroscope slide by unit gravity. When polymerization was complete,imaging was performed using an ArrayWoRx^(e) BioChip fluorescencemicroarray reader (Applied Precision, LLC, Issaquah, Wash.). The beadswere imaged in 2 different fluorescence channels to detect the Cy3 andCy5 hybridization probes.

Results:

Results show proof-of-principal for multiplexing MSP of multiple diseasebiomarkers, in this case for colorectal cancer, by using a singlesolid-phase bridge PCR reaction. FIG. 42 is a 2-color fluorescence imageoverlay of the solid-phase bridge PCR beads following dual hybridizationprobing for both vimentin (Cy3; green in FIG. 42) and RASSF2A (Cy5; redin FIG. 42) amplicons. In FIG. 42, panels marked as “Multiplex” pertainto where both vimentin and RASSF2A primer coated beads were included inthe solid-phase bridge PCR reaction at a 50:50 ratio. If only thetemplate DNA was omitted [“−Template (Multiplex)”], no significantsignal was observed. If both templates were included in the reaction[“+Vimentin & +RASSF2A (Multiplex)”], both amplicons were observed andwere segregated on their respective beads, with the two bead populationsin an approximate 50:50 ratio as expected. Controls where only oneprimer coated bead species and the corresponding template were used inthe solid-phase bridge PCR reaction show that only the respectiveamplicon was produced and detected (two right most panels in FIG. 42).

Example 48 Solid-Phase Bridge PCR on the APC Gene Associated withColorectal Cancer: Direct Use of Genomic DNA Templates in theSolid-Phase Bridge PCR Reaction

This Example illustrates 3 important aspects of the presentedsolid-phase bridge PCR methodology compared to previous Examples: i)All, rather than partial untranslated regions and epitope tag sequencesof the solid-phase bridge PCR amplicon are introduced by the solid-phasebridge PCR primers which allows ii) the direct use of native (i.e. noexogenous sequence modifications) genomic DNA templates, such as thoseobtained from patients, in the solid-phase bridge PCR reaction, and iii)the benefit of magnesium supplementation to improve the efficiency ofthe solid-phase bridge PCR reaction is demonstrated.

Preparing the Solid-Phase Bridge PCR Template DNA:

Note: All buffers and reagents used throughout this entire Example,unless otherwise noted, were minimally DNAse, RNAse and protease free,referred to as Molecular Biology Grade (MBG), including the water,referred to as MBG-Water.

Normal human blood genomic DNA (Clontech, Mountain View, Calif.) waspurchased commercially to be used as the template for solid-phase bridgePCR. The genomic DNA was first mechanically fragmented into an averagesize of roughly 500 bp via direct probe sonication. Fragmentation wasverified by standard agarose gel electrophoresis.

Preparation of Agarose Beads Covalently Conjugated to PCR Primers Usedfor Solid-Phase Bridge PCR:

Performed as in Example 36 with the following exceptions: PCR primersused in this Example are listed below this paragraph. In the primersbelow, the bracketed sequences indicate the gene-specific APC directedhybridization regions, while the remaining sequences are non-hybridizingregions which correspond to all of the elements needed for latercell-free protein expression as well as epitope tag detection. Duringconjugation to the beads, concentration of each primer was 29 μM insteadof 125 μM.

Solid-Phase Bridge PCR APC Forward Primer: [SEQ NO. 67]5′TAATACgACTCACTATAgggAgAggAggTATATCAATgTACACCgACATCgAgATgAACCgCCTgggCAAgggAggACAgCCTgAACTCgCTCCAgAggATCCggAAgAT[CAggAAgCAgATTCTgCTAAT]3′ Solid-Phase Bridge PCR APC ReversePrimer: [SEQ NO. 68] 5′TTTTTTTTTTTTTTTTTTTTATTATCCTCCTCCTTTATCATCATCgTCTTTATAATCCAgCAgCTTgTgCAggTCgCTgAAggT[TggACTTTTgggT gTCTgAgCACCACTTTT]3′

Qualitative Analysis of Primer Attachment:

Performed as in Example 36.

First Round of Effective Single Template Molecule Solid-Phase BridgePCR:

Performed essentially as in Example 36, with slight modifications. Thefull protocol was as follows: 4 μL actual bead volume of the previouslyprepared Primer-Conjugated Agarose Beads was used per each sample, butfirst, the beads were washed in bulk, with heating. To do so, 65 μL (induplicate) of the aforementioned 20% (v/v) Primer-Conjugated AgaroseBead suspension (˜13 μL actual bead volume) was placed into a 0.5 mLpolypropylene thin-wall PCR tube. The beads were spun down briefly in astandard micro-centrifuge (just until reaches maximum speed of ˜13,000rpm corresponding to ˜16,000×g). As much of the fluid supernatant wasremoved as possible by manual pipetting, with the beads nearly going todryness. 50 μL of TE-50 mM NaCl (10 mM Tris, pH 8.0, 1 mM EDTA, 50 mMNaCl) was added to the pellets, to bring the volume approximately backto the original 20% beads (v/v). The beads were briefly vortex mixedthen spun down and all fluid removed as described before. 50 μL of TE-50mM NaCl was again added to the pellets as above and the tubes placed ina PCR machine (Mastercycler Personal; Eppendorf AG, Hamburg, Germany) at95° C. for 10 min (lid temperature 105° C. and no mineral oil used)(beads were resuspended by brief gentle vortex mixing just before and at5 min of this step). After heating, the tube was immediately removedfrom the PCR machine, the beads diluted in 400 μL of TE-50 mM NaCl andthe bead suspension then transferred to a Filtration Device (see Example36). Filtration was performed and the filtrate discarded. Beads werebriefly washed 2×400 μL more with TE-50 mM NaCl then 2×400 μL withMBG-Water. Each set of beads was then resuspended in 50 μL MBG-Water andthen pooled. The pooled bead suspension was divided up into 0.5 mLpolypropylene thin-wall PCR tubes such that 4 μL actual bead volume wasadded per tube (i.e. per sample). The beads were spun down briefly in astandard micro-centrifuge (just until reaches maximum speed of ˜13,000rpm corresponding to ˜16,000×g). As much of the fluid supernatant wasremoved as possible by manual pipetting, with the beads nearly going todryness.

Next, to pre-hybridize the template DNA to the washed Primer-ConjugatedAgarose Beads, each 4 μL bead pellet was then resuspended in 2.5 μL ofdiluted fragmented genomic DNA template solution, which contained nosoluble primers. The aforementioned fragmented genomic DNA template,prepared as described in this Example, was diluted to 33.2 ng/μLdirectly in a commercially available pre-mixed PCR reaction solutioncontaining everything needed for PCR except template DNA and primers(Phusion™ High Fidelity PCR Master Mix; 1× contains 0.02 U/μL PhusionDNA Polymerase, 200 μM dNTPs, 1.5 mM MgCl₂ and other optimized bufferconstituents; New England Biolabs, Ipswich, Mass.; solution provided as2× concentrate and used at 1×). This resulted in a ratio of ˜3,000genome equivalents per μL of actual Primer-Conjugated Agarose Beadvolume. With 1 μL of Primer-Conjugated Agarose Beads determined tocontain approximately 1,000 beads, ˜3,000 genome equivalents per μL ofbeads represents a ratio of approximately 6 actual APC templatemolecules (gene copies) added per bead (beads physically enumeratedunder a microscope both in diluted droplets of bead suspension and withsuspensions in a hemacytometer cell counting chamber). It should benoted that although a ratio of 6 APC gene copies per bead was used, thefragmentation of the genomic DNA will statistically reduce the number ofamplifiable APC templates per bead (average genomic DNA templatefragment ˜500 bp; targeted APC region for amplification 237 bp). A minustemplate negative control was also prepared. The bead suspensions wereonly mixed manually by gentle stirring with a pipette tip.

The resultant bead suspensions, now containing added template but nosoluble (free) primers (only bead-bound primers), were then treated asfollows in a PCR machine (Mastercycler Personal; Eppendorf AG, Hamburg,Germany) (lid temperature 105° C., and no mineral oil used): 5 min 95°C. (denaturing), ramp down to 55° C. at a rate of 0.1° C./sec and thenhold 1 hour at 55° C. (annealing/capture of template onto beads). Next,30 μL of fresh 1× Phusion™ High Fidelity PCR Master Mix was added toeach sample (without removal of previous solution and without lettingsamples cool). At this stage, some samples also received magnesiumsupplementation beyond what was provided in the aforementioned 1×Phusion™ High Fidelity PCR Master Mix. The 3 sample permutations at thisstage were as follows: 1) Minus template; no supplementation 2) plustemplate; no supplementation 3) plus template; magnesium supplementationto 3 mM total (duplicate sample). Before adding the 30 μL solutions toeach sample, the solutions were pre-treated on a PCR machine at 98° C.for 3.7 min followed by 65° C. for 40 seconds; and the solutions thenadded while at 65° C. After addition of the solutions to the samples,the samples were treated at 72° C. for 10 min on the PCR machine tofully extend any primers which were hybridized to a template molecule.Immediately upon completion of the previous steps above, while the tubeswere still at 72° C., the tubes were immediately transferred from thePCR machine to a crushed ice water bath. 400 μL of ice cold MBG-Waterwas added to each tube, the suspensions transferred to fresh FiltrationDevices, filtration was immediately performed and the filtrate discarded(see Example 36). Using the same Filtration Devices, the beads werebriefly washed 2×400 μL with room temperature MBG-Water. Beads werefurther washed 2×400 μL for 3 min each with room temperature 0.1M NaOH,with constant vigorous vortex mixing, in order to strip off anyhybridized but non-covalently bound template DNA, leaving onlycovalently attached unused and extended primers on the beads. The beadswere then briefly washed 3×400 μL with 10×TE (100 mM Tris, pH 8.0, 10 mMEDTA), in order to neutralize the pH, followed by 3×400 μL withMBG-Water, in order to remove the components of the 10×TE which wouldinterfere with subsequent PCR.

Following the final filtration step on the bead samples, each washedbead pellet was resuspended in 50 μL of the aforementioned commercialPhusion™ High Fidelity PCR Master Mix pre-mixed PCR solution. Themagnesium supplementation detailed earlier was also maintained at thisstage for the corresponding samples. Also at this stage, some samplesadditionally received Phusion DNA polymerase supplementation beyond whatwas provided in the aforementioned 1× Phusion™ High Fidelity PCR MasterMix. The 4 sample permutations at this stage were as follows: 1) Minustemplate; no supplementation 2) plus template; no supplementation 3)plus template; magnesium supplementation to 3 mM total and 4) plustemplate; magnesium supplementation to 3 mM total with Phusion DNApolymerase supplementation to 0.1 U/μL total. The suspensions were thenrecovered from their Filtration Devices into fresh 0.5 mL polypropylenethin-wall PCR tubes and subjected to the following themmocycling in aPCR machine (Mastercycler Personal; Eppendorf AG, Hamburg, Germany) (lidtemperature 105° C. and no mineral oil used): An initial denaturing stepof 98° C. for 2 min (once) (beads were briefly resuspended by gentlevortex mixing just before this step), and 40 cycles of 98° C. for 40 sec(denature), 65° C. for 40 sec (anneal), and 72° C. for 1 min (extend);followed by a final extension step of 72° C. for 5 min (once).

400 μL of TE-50 mM NaCl (10 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl) wasadded to each completed solid-phase bridge PCR reactions and thesuspensions transferred to Filtration Devices (Ultrafree-MC DuraporeMicro-centrifuge Filtration Devices, 400 μL capacity; Millipore,Billerica, Mass.). Filtration was performed in a micro-centrifuge (justuntil reaches maximum speed of ˜13,000 rpm corresponding to ˜16,000×g)and the filtrate discarded. The beads were washed 2×400 μL more withTE-50 mM NaCl; resuspending by ˜5 sec vortex mixing then performingfiltration and discarding the filtrate as above. Beads were usedimmediately for a full second round of PCR thermocycling as describedbelow.

Second Round of Solid-Phase Bridge PCR:

A portion of the above beads, following completion of the aforementionedinitial full round of solid-phase bridge PCR thermocycling (i.e. allpreceding steps), were subjected to a second full round of PCRthermocycling. To do so, 2.5 μL actual bead volume was washed 3×400 μLwith MBG-Water using a Filtration Device. Following the final filtrationstep on the bead samples, each washed bead pellet was resuspended in 50μL of the aforementioned commercial Phusion™ High Fidelity PCR MasterMix pre-mixed PCR solution. The magnesium and Phusion DNA polymerasesupplementation detailed earlier was also maintained at this stage forthe corresponding samples. Therefore, the 4 sample permutations at thisstage remained as follows: 1) Minus template; no supplementation 2) plustemplate; no supplementation 3) plus template; magnesium supplementationto 3 mM total and 4) plus template; magnesium supplementation to 3 mMtotal with Phusion DNA polymerase supplementation to 0.1 U/μL total. Thesuspensions were then recovered from their Filtration Devices into fresh0.5 mL polypropylene thin-wall PCR tubes and subjected to the followingthermocycling in a PCR machine (Mastercycler Personal; Eppendorf AG,Hamburg, Germany) (lid temperature 105° C. and no mineral oil used): Aninitial denaturing step of 98° C. for 2 min (once) (beads were brieflyresuspended by gentle vortex mixing just before this step), and 40cycles of 98° C. for 40 sec (denature), 65° C. for 40 sec (anneal), and72° C. for 1 min (extend); followed by a final extension step of 72° C.for 5 min (once).

Next, the solid-phase bridge PCR product on the beads was hybridizedwith a fluorescently labeled complementary oligonucleotide directedagainst internal APC sequences. The oligonucleotide probe wascommercially custom synthesized with a 5′ Cy5 label and PAGE purified bythe manufacturer (Sigma-Genosys, The Woodlands, Tex.). The probe wasdiluted to 5 μM final in TE-50 mM NaCl for hybridization experiments.Prior to use however, the 5 μM probe solution was pre-clarified byspinning 1 min at maximum speed on a micro-centrifuge (˜13,000 rpm or˜16,000×g) and collecting the fluid supernatant. The supernatant wasthen passed though a Filtration Device (see earlier in this Example forFiltration Devices) and the filtrate saved for use as the probesolution. The sequence of the probe was as follows:

Internal APC Probe: 5′[Cy5]gCACCCTAgAACCAAATCCAgCAgACTg3′ [SEQ NO. 69]

To perform the hybridization probing, following completion of thesolid-phase bridge PCR reaction, 400 μL of TE-50 mM NaCl (10 mM Tris, pH8.0, 1 mM EDTA, 50 mM NaCl) was added to each completed solid-phasebridge PCR reaction and the suspensions transferred to fresh FiltrationDevices (Ultrafree-MC Durapore Micro-centrifuge Filtration Devices, 400μL capacity; Millipore, Billerica, Mass.). Filtration was performed andthe filtrate discarded. The beads were washed briefly 2×400 μL more withTE-50 mM NaCl. Prior to performing filtration on the final wash, enoughof the suspension was removed from the Filtration Device (for storage)thereby leaving 1 μL actual bead volume per sample. Followingfiltration, each 1 μL pellet corresponding to each sample wasresuspended in 25 μL of the aforementioned clarified 5 μM probesolution. The beads were resuspended by manual pipetting thentransferred to 0.5 mL polypropylene thin-wall PCR tubes. Hybridizationwas performed as follows in a PCR machine: 2 min 94° C. (denature)(beads resuspended by vortex mixing just before this step) followed byramping down to 68° C. at a rate of 0.1° C./sec and subsequently holding1 hour at 68° C. (anneal).

Just at the end of the above 1 hour 68° C. (anneal) step, while thetubes were still at 68° C. and still in the PCR machine, each sample wasrapidly diluted with 400 μL of 68° C. TE-50 mM NaCl, the suspensionsimmediately transferred to a Filtration Device and filtrationimmediately performed. The filtrate was then discarded. The beads werewashed 2×400 μL more with 68° C. TE-50 mM NaCl then 2×400 μL with roomtemperature TE-50 mM NaCl. Next, to fluorescently stain all beadsindependently of the presence or absence of amplicon, the beads weretreated 1× for 5 min with gentle mixing using 200 μL of TE-50 mM NaClcontaining 50 pg/μL of a streptavidin Alexa Fluor 488 conjugate(Invitrogen Corporation, Carlsbad, Calif.). The beads were then furtherwashed 2×400 μL with TE-50 mM NaCl and then 1× 400 μL with TE-100 mMNaCl (10 mM Tris, pH 8.0, 1 mM EDTA, 100 mM NaCl). The beads wererecovered from the Filtration Devices by resuspending the pellet in 50μL of TE-100 mM NaCl and transferring to a 0.5 mL polypropylene PCRtube. The beads were spun down in a standard micro-centrifuge (justuntil reaches maximum speed of ˜13,000 rpm corresponding to ˜16,000×g)and the fluid supernatant removed.

Embedding the Beads in a Polyacrylamide Film and Fluorescence Imaging:

Lastly, the beads were embedded in a polyacrylamide film on a microscopeslide and fluorescently imaged to detect the bound Cy5 labeledhybridization probe as well as the Alexa Fluor 488 total bead staining.To do so, an Acrylamide Mix was prepared by combining the followingreagents in order: 244 μL of TE-100 mM NaCl, 57 μL of 40% acrylamide(19:1 cross-linking) (Bio-Rad Laboratories, Hercules, Calif.), 0.5 μLTEMED (Bio-Rad Laboratories, Hercules, Calif.), and 1 μL of a 10% (w/v)ammonium persulfate stock (prepared in MBG-Water from powder obtainedfrom Bio-Rad Laboratories, Hercules, Calif.). Each aforementioned washedbead pellet was then resuspended in 50 μL of the above Acrylamide Mixand combined by brief vortex mixing. 25 μL of the bead suspension wasthen pipetted to a standard glass microscope slide and overlaid with astandard 18 mm square microscope cover glass (coverslip). Polymerizationwas allowed to occur for ˜10 min protected from light. Note that theadequately slow polymerization process allows all beads to settle to thesurface of the microscope slide by unit gravity. When polymerization wascomplete, imaging was performed using an ArrayWoRx^(e) BioChipfluorescence microarray reader (Applied Precision, LLC, Issaquah,Wash.).

Results:

The fluorescence images are shown in FIG. 43 as 2-color overlays. Thegreen signal in FIG. 43 is the total bead stain, independent of thepresence or absence of amplicon, while the red signal is theAPC-specific hybridization probing of the solid-phase bridge PCRamplicon on the beads. Without magnesium supplementation in thesolid-phase bridge PCR reaction, essentially no amplicon is detectibleon the beads above the minus template background control sample. Withmagnesium supplementation to 3 mM total, solid-phase bridge PCR amplicon(APC) is detected in the plus template sample permutation, with asignal-to-noise ratio of approximately 5:1 when quantified. 3 mM totalmagnesium plus DNA polymerase supplementation to 0.1 U/μL furtherimproves solid-phase bridge PCR efficiency approximately 2-3 fold abovethe 3 mM magnesium alone. These results confirm the compatibility of thepresented solid-phase bridge PCR method with amplification of fragmentedgenomic DNA templates, and also demonstrates the benefits of magnesiumsupplementation in the solid-phase bridge PCR reaction. Magnesiumsupplementation is beneficial likely due to the high concentration ofprimers on the beads, which chelate the magnesium thereby reducing thefree magnesium concentration in the reaction. Sufficient free magnesiumhowever, is needed as a co-factor for the DNA polymerase activity.

Example 49 Solid-Phase Bridge PCR on 6 Micron Diameter, Non-Porous,Fluorescently Bar-Coded Plastic Beads from Luminex Corporation:Detection of the Solid-Phase Bridge PCR Amplicon on the Beads byBiotin-dUTP Labeling

This Example demonstrates the compatibility of solid-phase bridge PCRwith multiplexed assay platforms, more specifically, the Luminex xMAP®platform (Luminex Corporation; Austin, Tex.) which currently can useapproximately 100 different fluorescently bar-coded bead species formultiplexed assays based on a flow cytometric readout.

Primer Conjugation to Luminex Beads

Note: All buffers and reagents used throughout this entire Example,unless otherwise noted, were minimally DNAse, RNAse and protease free,referred to as Molecular Biology Grade (MBG), including the water,referred to as MBG-Water.

xMAP Multi-Analyte Carboxylated Microspheres® and SeroMAP CarboxylatedMicrospheres® were purchased commercially from Luminex Corporation(Austin, Tex.). The beads are non-porous, polystyrene based, containcarboxyl functional moieties and have a diameter of approximately 6microns.

These carboxylated beads were covalently conjugated to the 5′ aminemodified solid-phase bridge PCR primers. The forward and reversesolid-phase bridge PCR primers, directed against a prepared templatecorresponding to a segment of the human APC gene Mutation Cluster Region(MCR), were purchased from Sigma-Genosys (The Woodlands, Tex.), bothwith a 5′ primary amine modification following a 6 carbon spacer. Theprimer sequences are listed below. In the primers below, the bracketedsequences indicate the template-specific hybridization regions, whilethe remaining sequences are non-hybridizing regions which correspond tothe remaining portions of the elements needed for later cell-freeprotein expression as well as epitope tag detection (the initial portionof these elements was introduced during the template preparation;template prepared as in Example 44).

Solid-Phase Bridge PCR APC Forward Primer: [SEQ NO. 70]5′[Amine]TAATACgACTCACTATAgggAgAggAggTATATCAATgTACACCgACATCgAg[ATgAACCgCCTgggCAAgggAggAggAggA]3′ Solid-Phase Bridge PCRAPC Reverse Primer: [SEQ NO. 71]5′[Amine]TTTTTTTTTTTTTTTTTTTTATTATCCTCCTCCTgCgTAgTCTggTACgTCgTATgggTA[CAgCAgCTTgTgCAggTCgCTgAAggTg g]3′

To wash and manipulate the beads or exchange the buffers, 0.45 micronpore size, PVDF membrane, micro-centrifuge Filtration Devices were usedunless otherwise noted (Ultrafree-MC Durapore Micro-centrifugeFiltration Devices, 400 μL capacity; Millipore, Billerica, Mass.). Usingthe aforementioned Filtration Devices, 5 μL of actual bead volume waswashed 5×400 μL with MES Buffer (0.1 M MES, pH 4.7, 0.9% NaCl; PierceBiotechnology, Inc., Rockford, Ill.). Unless otherwise noted, all washesare brief, 1-3 sec, by vortex mixing followed by spinning the FiltrationDevices briefly in a standard micro-centrifuge (just until reachesmaximum speed of ˜13,000 rpm corresponding to ˜16,000×g) and discardingthe filtrate. The washed bead pellets were then recovered into 0.5 mLpolypropylene PCR tubes by resuspending in 120 μL of MES Buffer. Eachsuspension was then split into 20 μL and 100 μL portions for the minusprimer and plus primer permutations respectively (roughly 1 μL and 4 μLactual bead volumes respectively). To the 100 μL bead suspensions, 5.1μL of a solution of 625 μM each primer (forward and reverse; prepared inMBG-Water) was added, resulting in a final concentration of 30 μM eachprimer (forward and reverse) (plus primer permutation). Nothing wasadded to the 20 μL bead suspensions (minus primer permutation).

EDC (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride powder;Pierce Biotechnology, Inc., Rockford, Ill.) was dissolved to 100 mg/mLin ice-cold MBG-Water then 5 μL and 25 μL immediately added to the aboveminus primer and plus primer bead suspensions respectively. The reactionwas carried out for 1 hour at room temperature with gentle mixing.

In Filtration Devices, the beads were then washed 3×400 μL withTE-Saline-Glycine Buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 200 mMNaCl, 0.1M glycine) and quenched by treatment for 30 min with mixing ina fresh 400 μL of the same buffer. Beads were then washed 4×400 μL withTE-50 mM NaCl-T Buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 50 mM NaCl,0.01% Tween-20) and lastly resuspended to 5% beads (v/v) in the samebuffer for storage at +4° C. protected from light.

Qualitative Analysis of Primer Attachment:

To qualitatively verify successful primer attachment to the beads, analiquot of the beads was stained with the single-stranded DNAfluorescence-based detection reagent OliGreen (Invitrogen Corporation,Carlsbad, Calif.). The manufacturer supplied reagent was diluted 1/200in TE (10 mM Tris, pH 8.0, 1 mM EDTA) containing 0.01% (v/v) Tween-20.2.5 μL of the prepared primer-conjugated bead suspension (5% beads for0.125 μL actual bead volume) was mixed with 100 μL of the dilutedOliGreen reagent in a thin-walled 0.5 mL clear polypropylene PCR tube.As a negative control, the beads that were prepared in the same manner,except lacked any attached primer, were also tested. After approximately1 min, the beads were spun down briefly in a micro-centrifuge (justuntil reaches maximum speed of ˜13,000 rpm corresponding to ˜16,000×g)and the bead pellet imaged directly in the tubes using a laser-basedfluorescence scanner (FUJI FLA-2000, 473 nm solid-state laser excitationand 520 nm emissions filter) (FUJI Photo Film Co. Ltd, Japan).

Template Preparation and Solid-Phase Bridge PCR

Template was prepared and purified as described in Example 44.

0.25 μL of actual bead volume of each of the 2 primer bead types (xMAPand SeroMAP) was washed in bulk in the aforementioned Filtration Devices1×400 μL with MBG-Water. Only beads containing primer were used in thesolid-phase bridge PCR reactions. Each bead type was then resuspended in400 μL of 0.1% (w/v) nuclease-free BSA (Invitrogen Corporation,Carlsbad, Calif.), in the top chamber of the Filtration Device, andallowed to stand for 15 min. Filtration was then performed and each beadpellet resuspended in 55 μL of PCR Master Mix (SuperTaq™ DNA PolymeraseKit; Ambion, Austin, Tex.; prepared according to the manufacturer'sinstructions except with 0.25 U/μL final SuperTaq™ DNA polymerase and 5%v/v PCR grade DMSO). Biotin-16-dUTP (Roche Applied Science,Indianapolis, Ind.) was also included in the PCR Master Mix at a finalconcentration of 20 μM, in order to label the solid-phase bridge PCRamplicon. Each bead suspension was then split into two 25 μL portionsinto 0.5 mL thin-wall polypropylene PCR tubes for the minus template andplus template sample permutations (approximately 0.125 μL/tube actualbead volume for approximately 300,000 beads/tube). For the plus templatesample permutations, 4 ng in 1 μL of the aforementioned template wasadded to the appropriate bead suspensions. Minus template samplepermutations received nothing further. The bead suspensions weresubjected to thermocycling as follows: Initially 94° C. 2 min (once);then 35 cycles of 94° C. 30 s, 65° C. 30 s and 72° C. 2 min; followed bya final 72° C. 10 min (once). Beads were resuspended by vortex mixingjust before thermocycling and then periodically every 5 cycles duringthe 72° C. 2 min extension step. After thermocycling, beads were washeddirectly in their tubes 5×400 μL with TE-50 mM NaCl-T Buffer. Beads wereultimately resuspended in approximately 25 μL of TE-50 mM NaCl-T Bufferfor storage at +4° C.

Chemiluminescence Based Detection of Biotin dUTP Labeled Amplicon onBeads

The above beads were blocked 30 min with gentle mixing by adding 400 μLof Blocking Buffer [1% (w/v) nuclease-free BSA in 10 mM Tris-HCl, pH8.0, 1 mM EDTA, 200 mM NaCl, 0.05% (v/v) Tween-20]. Beads were spun downbriefly in a standard micro-centrifuge (just until reaches maximum speedof ˜13,000 rpm corresponding to ˜16,000×g) and the supernatantdiscarded. Beads were then treated with 200 μL of 50 ng/mL of aNeutrAvidin-HRP conjugate (Pierce Biotechnology, Inc., Rockford, Ill.)diluted in Blocking Buffer. Treatment was for 30 min with gentle mixing.Beads were then spun down briefly in a standard micro-centrifuge (justuntil reaches maximum speed of ˜13,000 rpm corresponding to ˜16,000×g)and the supernatant discarded. Beads were washed 4×400 μL inTE-Saline-Tween (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 200 mM NaCl, 0.05%(v/v) Tween-20). All washes were by brief (1-3 sec) vortex mixingfollowed by spinning the beads down briefly in a standardmicro-centrifuge (just until reaches maximum speed of ˜13,000 rpmcorresponding to ˜16,000×g) and discarding the supernatant. Beads werethen resuspended in 400 μL of Tris-Saline (10 mM Tris-HCl, pH 8.0, 200mM NaCl) and transferred to the aforementioned Filtration Devices (freshdevices). Filtration was performed as before and the filtrate discarded.Beads were then recovered from the Filtration Devices in 50 μL ofTris-Saline and placed into the wells of a 96-well opaque whitemicrotiter plate. Next, 200 μL/well was added of freshly preparedSuperSignal Femto chemiluminescent HRP substrate (Pierce Biotechnology,Inc., Rockford, Ill.), the plates shaken for 10 s and immediately readon a LumiCount luminescence plate reader (Packard/PerkinElmer Life andAnalytical Sciences, Inc., Boston, Mass.).

Results:

Primer attachment to the beads was first verified by staining the beadswith OliGreen, which fluorescently detects single-stranded DNA. Thestained bead pellets were imaged directly in 0.5 mL thin-wallpolypropylene PCR tubes and the image shown in FIG. 44A. The resultsclearly show that attached primer is only detected when the primers wereincluded in the chemical conjugation reaction (“+Primer”), but not whenthe primers were omitted from the reaction (“−Primer”).

Following verification or primer attachment, the beads (not stained withOliGreen) were used in solid-phase bridge PCR reactions (only beadscontaining primer used in solid-phase bridge PCR). The resultantamplicon on the beads, which was labeled using biotin dUTP during thesolid-phase bridge PCR, was detected via a chemiluminescent assay. Thedata was plotted and is shown graphically in FIG. 44B. Results show thatsolid-phase bridge PCR amplicon is clearly detected on both the xMAP andSeroMAP beads only when the template DNA is added to the solid-phasebridge PCR reaction, with signal-to-noise ratios of 133:1 and 250:1respectively.

Example 50 Solid-Phase Bridge PCR for Detection of the BisulfiteConverted Wild-Type Vimentin DNA Marker Directly from Genomic DNA:Applications in Colorectal Cancer Diagnosis

Methylation of the vimentin and RASSF2A markers and the detection ofcolorectal cancer, using methylation-specific PCR (MSP), is reported inthe scientific literature [Chen et al. (2005) J Natl Cancer Inst 97,1124-1132; Hesson et al. (2005) Oncogene 24, 3987-3994; Park et al.(2007) Int J Cancer 120, 7-12]. This Example demonstrates the ability touse solid-phase bridge PCR for MSP assays on diagnostic markers. In thisExample, detection of the wild-type vimentin marker is demonstrated.However, the technique is equally applicable to the detection of the“mutant”, i.e. methylated marker(s), simply by changing the primersequences.

Importantly, this example differs from Example 47 in that fragmented andbisulfite converted genomic DNA was used directly as the solid-phasebridge PCR (MSP) template, instead of a purified PCR product as donepreviously.

Preparation of Agarose Beads Covalently Conjugated to PCR Primers Usedfor Solid-Phase Bridge PCR:

Production of Primer-Conjugated Agarose Beads was performed as inExample 36 except beads with the following primer pair were prepared.

Vimentin Unmethylated Primer Pair: [SEQ NO. 72] Forward:5′[Amine]TTgAggTTTTTgTgTTAgAgATgTAgTTgT3′ [SEQ NO. 73] Reverse:5′[Amine]ACTCCAACTAAAACTCAACCAACTCACA3′

Qualitative Analysis of Primer Attachment:

Performed as in Example 36

Template for Solid-Phase Bridge PCR and Bisulfite Conversion:

Normal human blood genomic DNA (wild-type DNA; i.e. unmethylated atvimentin marker region) (Clontech, Mountain View, Calif.) was purchasedcommercially to be used as the template for solid-phase bridge PCR. Thegenomic DNA was first mechanically fragmented into an average size ofroughly 500 bp via direct probe sonication. Fragmentation was verifiedby standard agarose gel electrophoresis.

For bisulfite conversion, the fragmented normal human blood genomic DNAwas first denatured by preparing the following reaction: 12.5 ng/μLsingle-stranded carrier DNA (lambda DNA, E. coli genomic DNA or salmonsperm DNA), 0.3N NaOH, and 1-50 ng of the aforementioned normal humanblood genomic DNA. The denaturation reaction was then incubated for 10min at 37° C. Next, 30 μL of 10 mM hydroquinone was added (10 mMhydroquinone prepared fresh from 25× stock which is stored at −20° C.)followed by 500 μL of a 3M sodium bisulfite stock (stock adjusted to pH5.0 with NaOH). Lastly, 200 μL of mineral oil was added and the reactionincubated at 50° C. for 16 hrs.

The resultant bisulfite converted DNA was purified using thecommercially available Wizard® DNA Clean-Up System (Promega, Madison,Wis.) according to the manufacturer's instructions. After elution fromthe Wizard® DNA Clean-Up System mini-columns in 90 μL 0.1×TE (1 mMTris-HCl, pH 8.0, 0.1 mM EDTA), the DNA was ethanol precipitated.Ethanol precipitation was carried out as follows: 45 μL of 1 N NaOH wasadded to each sample and briefly vortex mixed. After 5 min, 15 μL of 3Msodium acetate, pH 5.2, was added to each tube. Next, 1 μL of 20 mg/mLglycogen was added followed by 300 μL of ethanol. The mixture was thenincubated at −80° C. for 20 min and spun in a micro-centrifuge for 10min (maximum speed of ˜13,000 rpm corresponding to ˜16,000×g). Theethanol was removed and the DNA pellet air dried at room temperature for15 min. The DNA was then re-dissolved in 0.1×TE for immediate use orstorage at −20° C. This fragmented and bisulfite converted genomic DNAdirectly served as template for the solid-phase bridge PCR reactionsdescribed below.

Solid-Phase Bridge PCR:

10 μL actual total bead volume of the previously preparedPrimer-Conjugated Agarose Beads was pre-washed in bulk. Beads werepre-washed to remove any non-covalently attached primers. Beads wereinitially washed using 0.45 micron pore size, PVDF membrane,micro-centrifuge Filtration Devices (Ultrafree-MC DuraporeMicro-centrifuge Filtration Devices, 400 μL capacity; Millipore,Billerica, Mass.). Unless otherwise noted, all washes involving theFiltration Devices were by brief vortex mixing (˜5 sec), spinning downbriefly in a micro-centrifuge (just until reaches maximum speed of˜13,000 rpm corresponding to ˜16,000×g) and discarding the filtrate.Initial washes were 2×400 μL with TE-50 mM NaCl (10 mM Tris, pH 8.0, 1mM EDTA, 50 mM NaCl). Beads were then resuspended in TE-50 mM NaCl to20% (v/v) and the suspensions recovered into 0.5 mL thin-walledpolypropylene PCR tubes. The tubes were placed in a PCR machine at 95°C. for 10 min to allow heat-mediated washing (lid temperature 105° C.and no mineral oil used) (beads were resuspended by brief gentle vortexmixing just before this step). After heating, the tubes were immediatelyremoved from the PCR machine, the beads were diluted to 400 μL withTE-50 mM NaCl and the bead suspensions were then transferred toFiltration Devices. Filtration was performed and the filtrate discarded.Beads were briefly washed 1×400 μL more with TE-50 mM NaCl then 1×400 μLwith MBG-Water.

Prior to the final filtration step (wash), bead suspensions were splitinto 2.5 μL and 7.5 μL portions (actual bead volume) in separateFiltration Devices. Following the final filtration step (wash) on thebead samples, the washed bead pellets were resuspended in a commerciallyavailable PCR reaction buffer (HotStarTaq DNA Polymerase kit; Qiagen,Valencia, Calif.). To do so, first, the 2.5 and 7.5 μL bead pellets wereresuspended in 100 μL each of the 1× HotStarTaq reaction buffer (i.e.just the provided buffer; no dNTPs or DNA polymerase yet) containingeither no template or roughly 150 ng of the aforementioned fragmentedand bisulfite converted genomic DNA template, respectively. Thisresulted in a ratio of ˜3,000 genome equivalents per μL of actualPrimer-Conjugated Agarose Bead volume. With 1 μL of Primer-ConjugatedAgarose Beads determined to contain approximately 1,000 beads, ˜3,000genome equivalents per μL of beads represents a ratio of approximately 6actual vimentin template molecules (gene copies) added per bead (beadsphysically enumerated under a microscope both in diluted droplets ofbead suspension and with suspensions in a hemacytometer cell countingchamber). It should be noted that although a ratio of 6 vimentin genecopies per bead was used, the fragmentation of the genomic DNA willstatistically reduce the number of amplifiable vimentin templates perbead (average genomic DNA template fragment ˜500 bp; targeted vimentinregion for amplification 217 bp). The suspensions were placed into 0.5mL polypropylene thin-wall PCR tubes and mixed at 57° C. for 18 hrs toselectively capture the targeted vimentin template by hybridization tothe bead-bound primers. Using the aforementioned Filtration Devices, thebeads were then washed 2×400 μL with TE-50 mM NaCl then 1×400 μL withthe 1× HotStarTaq reaction buffer (i.e. just the provided buffer; nodNTPs or DNA polymerase yet) to remove any unbound DNA. Then, 2.5 μLactual bead volume, from each sample, was each resuspended in 25 μL ofHotStarTaq DNA Polymerase PCR reaction mix which as prepared accordingto the manufacturer's instructions (with dNTPs and DNA polymerase atthis stage) and pre-activated (95° C. for 15 min then cool to roomtemperature) prior to addition to the beads. The samples were thensubjected to the following thermocycling in a PCR machine (lidtemperature 105° C. and no mineral oil used): An initial extension step(once) of 72° C. for 10 min followed by an initial denaturing step(once) of 95° C. for 5 min, and 70 cycles of 94° C. for 30 sec(denature), 58° C. for 2 min (anneal) and 72° C. for 1 min (extend)whereby a fresh aliquot of HotStarTaq DNA polymerase was added to 0.05U/μL final concentration after 40 cycles; followed by a final extensionstep of 72° C. for 5 min after all 70 cycles.

400 μL of TE-50 mM NaCl (10 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl) wasadded to each completed solid-phase bridge PCR reaction and thesuspensions transferred to Filtration Devices (Ultrafree-MC DuraporeMicro-centrifuge Filtration Devices, 400 μL capacity; Millipore,Billerica, Mass.). Filtration was performed in a micro-centrifuge (justuntil reaches maximum speed of ˜13,000 rpm corresponding to ˜16,000×g)and the filtrate discarded. The beads were washed 3×400 μL more withTE-50 mM NaCl; resuspending by ˜5 sec vortex mixing then performingfiltration and discarding the filtrate as described earlier in thisExample.

Oligonucleotide Hybridization Probing:

Fluorescently labeled oligonucleotide probes were commercially customsynthesized and HPLC purified by the manufacturer (Sigma-Genosys, TheWoodlands, Tex.). The probes were reconstituted to 100 μM in MBG-Waterand further desalted using MicroSpin G-25 columns according to themanufacturer's instructions (Amersham Biosciences Corp., Piscataway,N.J.), except that the columns were pre-washed 2×350 μL with MBG-Waterprior to sample loading (to wash, columns were mixed briefly in theMBG-Water then spun 1 min in a standard micro-centrifuge at the properspeed). The probes were diluted in TE-50 mM NaCl for hybridizationexperiments. Prior to use however, the diluted probe solution waspre-clarified by spinning 1 min at maximum speed on a micro-centrifuge(˜13,000 rpm or ˜16,000×g) and collecting the fluid supernatant. Thesupernatant was then passed though a Filtration Device (see earlier inthis Example for Filtration Devices) and the filtrate was saved for useas the clarified probe solution.

In this Example, hybridization probing was performed by creating aprobing solution containing 1 μM of the vimentin probe, labeled on its5′ end with the Cy3 fluorophore by the manufacturer (Sigma-Genosys, TheWoodlands, Tex.). The gene-specific probe was complementary to aninternal segment of the vimentin solid-phase bridge PCR amplicon:

Human Vimentin Unmethylated & Bisulfite Converted:5′[Cy3]TgTAggATgTTTggTggTTTggg3′ [SEQ NO. 74]

After solid-phase bridge PCR and washing of the beads as describedearlier in this Example, the bead pellets corresponding to each samplewere resuspended in 25 μL of the aforementioned clarified probesolution. The beads were resuspended by manual pipetting thentransferred to 0.5 mL polypropylene thin-wall PCR tubes. Hybridizationwas performed as follows in a PCR machine (lid temperature always 105°C., no mineral oil used): 5 min 95° C. (denature) (beads were beresuspended by vortex mixing just before this step) followed by rampingdown to 60° C. at a rate of 0.1° C./sec and subsequently holding 1 hourat 60° C. (anneal).

Just at the end of the above 1 hour 60° C. (anneal) step, while thetubes were still at 60° C. and still in the PCR machine, each sample wasrapidly diluted with 400 μL of 60° C. TE-50 mM NaCl, the suspensionsimmediately transferred to a Filtration Device and filtrationimmediately performed. The filtrate was then discarded. The beads werewashed 2×400 μL more with 60° C. TE-50 mM NaCl then 1×400 μL with roomtemperature TE-50 mM NaCl. Beads were lastly washed 1×400 μL with TE-100mM NaCl (10 mM Tris, pH 8.0, 1 mM EDTA, 100 mM NaCl). The beads wererecovered from the Filtration Devices by resuspending the pellets in 50μL of TE-100 mM NaCl and transferring to a 0.5 mL polypropylene PCRtube. The beads were spun down in a standard micro-centrifuge (justuntil reaches maximum speed of ˜13,000 rpm corresponding to ˜16,000×g)and the fluid supernatant was removed.

Embedding the Beads in a Polyacrylamide Film and Fluorescence Imaging:

Lastly, the beads were embedded in a polyacrylamide film on a microscopeslide and fluorescently imaged to detect the bound Cy3 labeledhybridization probe. To do so, an Acrylamide Mix was prepared bycombining the following reagents in order: 244 μL of TE-100 mM NaCl, 57μL of 40% acrylamide (19:1 cross-linking) (Bio-Rad Laboratories,Hercules, Calif.), 0.5 μL TEMED (Bio-Rad Laboratories, Hercules,Calif.), and 1 μL of a 10% (w/v) ammonium persulfate stock (prepared inMBG-Water from powder obtained from Bio-Rad Laboratories, Hercules,Calif.). Each aforementioned washed bead pellet was then resuspended to2% (v/v) beads in the above Acrylamide Mix and combined by brief vortexmixing. 25 μL of the bead suspension was then pipetted to a standardglass microscope slide and overlaid with a standard 18 mm squaremicroscope cover glass (coverslip). Polymerization was allowed to occurfor ˜10 min protected from light. Note that the adequately slowpolymerization process allows all beads to settle to the surface of themicroscope slide by unit gravity. When polymerization was complete,imaging was performed using an ArrayWoRx^(e) BioChip fluorescencemicroarray reader (Applied Precision, LLC, Issaquah, Wash.).

Results:

Results show proof-of-principal for performing methylation-specific PCR(MSP) on disease biomarkers (in this case for colorectal cancer), usingsolid-phase bridge PCR, by directly using fragmented and bisulfiteconverted genomic DNA as the template. FIG. 45 shows the solid-phasebridge PCR beads following fluorescence hybridization probing for thevimentin amplicon. Amplicon is detected on the beads when the fragmentedand bisulfite converted genomic DNA template is added to the solid-phasebridge PCR reaction (“+gDNA Template”), with a signal-to-noise ratio ofapproximately 10:1 (following quantification) versus the negativecontrol sample where only the template DNA was omitted (“−Template”).

Example 51 Effective Single Template Molecule Solid-Phase Bridge PCR onthe APC Gene Associated with Colorectal Cancer: Multiplexing of VariousAPC Gene Segments Using Patient DNA as Template and Followed by aDownstream Bead-Based High-Sensitivity Protein Truncation Test

The overall goal of this Example is molecular diagnostics of colorectalcancer based on detection of truncating mutations in the APC gene fromvarious types of patient samples (e.g. stool, urine or blood samples).These patient samples are the source of template DNA molecules used inthe solid-phase bridge PCR and cell-free protein expression baseddiagnostic test. This Example will combine the effective amplificationof single template DNA molecules per bead in solid-phase bridge PCR,e.g. similar to as in Example 44, with the inherent multiplexingcapabilities of solid-phase bridge PCR (e.g. Example 47), that is, touse different primer bead species in a single reaction to target(amplify) single molecules from different segments of the APC geneassociated with colorectal cancer. Following solid-phase bridge PCR,multiplexed cell-free expression, with in situ capture, e.g. as inExample 31, will be performed on the bead population to convert the DNAbeads to beads carrying the cognate protein. Lastly, the beads, orbead-derived contact photo-transfer spots, will be probed withfluorescently labeled antibodies to N-terminal and C-terminal detectionepitopes to measure the presence of beads (or bead-derived spots)carrying truncated proteins, e.g. as in Example 42, indicating thepresence of a truncation mutation in APC in at least a fraction of thetemplate DNA molecules from the patient sample.

Solid-Phase Bridge PCR Template DNA:

DNA will be isolated from various biological fluids and biologicalsamples from both normal human subjects as well as those known to havecolorectal cancer at various stages (e.g. as identified by colonoscopy).Fluids and samples will include, but are not limited to tissue, stool,blood, serum, plasma or urine.

Preparation of Agarose Beads Covalently Conjugated to PCR Primers Usedfor Solid-Phase Bridge PCR:

Several different Primer-Conjugated Agarose Bead sets will be preparedseparately, each set targeting different regions of Exon 15 of the APCgene, with all sets combined covering the entire Mutation Cluster Region(MCR). Primer-Conjugated Agarose Bead preparation will be performed asin Example 48, except using the PCR primer pairs listed below thisparagraph. In the primers below, the bracketed sequences indicate thegene-specific APC directed hybridization regions, while the remainingsequences are non-hybridizing regions which correspond to all of theelements needed for later cell-free protein expression as well asepitope tag binding and detection. Epitope tags include an N-terminalVSV-G detection tag, an N-terminal HSV capture/binding tag and aC-terminal p53-tag for detection. For the reference APC templatesequence (mRNA), see for example GeneBank Accession NM_(—)000038.

APC Segment 1 (Forward and Reverse): [SEQ NO. 75]5′TAATACgACTCACTATAgggAgAggAggTATATCAATgTACACCgACATCgAgATgAACCgCCTgggCAAgggAggACAgCCTgAACTCgCTCCAgAggATCCggAAgAT[ggACAAAgCAgTAAAACCgAA]3′ [SEQ NO. 76]5′TTATTACAgCAgCTTgTgCAggTCgCTgAAggT[AgCCTTTTgAggCT gACCACT]3′ APCSegment 2 (Forward and Reverse): [SEQ NO. 77]5′TAATACgACTCACTATAgggAgAggAggTATATCAATgTACACCgACATCgAgATgAACCgCCTgggCAAgggAggACAgCCTgAACTCgCTCCAgAggATCCggAAgAT[CCAAgTTCTgCACAgAgTAgA]3′ [SEQ NO. 78]5′TTATTACAgCAgCTTgTgCAggTCgCTgAAggT[TgAACTACATCTTg AAAAACA]3′ APCSegment 3 (Forward and Reverse): [SEQ NO. 79]5′TAATACgACTCACTATAgggAgAggAggTATATCAATgTACACCgACATCgAgATgAACCgCCTgggCAAgggAggACAgCCTgAACTCgCTCCAgAggATCCggAAgAT[TgTgTAgAAgATACTCCAATA]3′ [SEQ NO. 80]5′TTATTACAgCAgCTTgTgCAggTCgCTgAAggT[TATTTCTgCTATTT gCAgggT]3′ APCSegment 4 (Forward and Reverse): [SEQ NO. 81]5′TAATACgACTCACTATAgggAgAggAggTATATCAATgTACACCgACATCgAgATgAACCgCCTgggCAAgggAggACAgCCTgAACTCgCTCCAgAggATCCggAAgAT[CAggAAgCAgATTCTgCTAAT]3′ [SEQ NO. 82]5′TATTACAgCAgCTTgTgCAggTCgCTgAAggT[CTgCAgTCTgCTggA TTTggT]3′ APC Segment5 (Forward and Reverse): [SEQ NO. 83]5′TAATACgACTCACTATAgggAgAggAggTATATCAATgTACACCgACATCgAgATgAACCgCCTgggCAAgggAggACAgCCTgAACTCgCTCCAgAggATCCggAAgAT[gCAgTgTCACAgCACCCTAgA]3′ [SEQ NO. 84]5′TTATTACAgCAgCTTgTgCAggTCgCTgAAggT[gggTgTCTgAgCAC CACTTTT]3′ APCSegment 6 (Forward and Reverse): [SEQ NO. 85]5′TAATACgACTCACTATAgggAgAggAggTATATCAATgTACACCgACATCgAgATgAACCgCCTgggCAAgggAggACAgCCTgAACTCgCTCCAgAggATCCggAAgAT[TCAggAgCgAAATCTCCCTCC]3′ [SEQ NO. 86]5′TTATTACAgCAgCTTgTgCAggTCgCTgAAggT[CgAACgACTCTCAA AACTATC]3′ APCSegment 7 (Forward and Reverse): [SEQ NO. 87]5′TAATACgACTCACTATAgggAgAggAggTATATCAATgTACACCgACATCgAgATgAACCgCCTgggCAAgggAggACAgCCTgAACTCgCTCCAgAggATCCggAAgAT[TgTACTTCTgTCAgTTCACTT]3′ [SEQ NO. 88]5′TTATTACAgCAgCTTgTgCAggTCgCTgAAggT[CATggTTTgTCCAg ggCTATC]3′ APCSegment 8 (Forward and Reverse): [SEQ NO. 89]5′TAATACgACTCACTATAgggAgAggAggTATATCAATgTACACCgACATCgAgATgAACCgCCTgggCAAgggAggACAgCCTgAACTCgCTCCAgAggATCCggAAgAT[ATAAgCCCCAgTgATCTTCCA]3′ [SEQ NO. 90]5′TTATTACAgCAgCTTgTgCAggTCgCTgAAggT[CTTTTCAgCAgTAg gTgCTTT]3′ APCSegment 9 (Forward and Reverse): [SEQ NO. 91]5′TAATACgACTCACTATAgggAgAggAggTATATCAATgTACACCgACATCgAgATgAACCgCCTgggCAAgggAggACAgCCTgAACTCgCTCCAgAggATCCggAAgAT[AAgCgAgAAgTACCTAAAAAT]3′ [SEQ NO. 92]5′TTATTACAgCAgCTTgTgCAggTCgCTgAAggT[CgTggCAAAATgTA ATAAAgT]3′ APCSegment 10 (Forward and Reverse): [SEQ NO. 93]5′TAATACgACTCACTATAgggAgAggAggTATATCAATgTACACCgACATCgAgATgAACCgCCTgggCAAgggAggACAgCCTgAACTCgCTCCAgAggATCCggAAgAT[CAggTTCTTCCAgATgCTgAT]3′ [SEQ NO. 94]5′TTATTACAgCAgCTTgTgCAggTCgCTgAAggT[TATTCTTAATTCCA CATCTTT]3′ APCSegment 11 (Forward and Reverse): [SEQ NO. 95]5′TAATACgACTCACTATAgggAgAggAggTATATCAATgTACACCgACATCgAgATgAACCgCCTgggCAAgggAggACAgCCTgAACTCgCTCCAgAggATCCggAAgAT[CTCgATgAgCCATTTATACag]3′ [SEQ NO. 96]5′TTATTACAgCAgCTTgTgCAggTCgCTgAAggT[TTTTTCTgCCTCTT TCTCTTg]3′ APCSegment 12 (Forward and Reverse): [SEQ NO. 97]5′TAATACgACTCACTATAgggAgAggAggTATATCAATgTACACCgACATCgAgATgAACCgCCTgggCAAgggAggACAgCCTgAACTCgCTCCAgAggATCCggAAgAT[CCTAAAgAATCAAATgAAAAC]3′ [SEQ NO. 98]5′TTATTACAgCAgCTTgTgCAggTCgCTgAAggT[TgACTTTgTTggCA TggCAgA]3′

Qualitative Analysis of Primer Attachment:

Will be performed as in Example 36.

First Round of Effective Single Template Molecule Solid-Phase BridgePCR:

Will be performed as in Example 48, using the conditions correspondingto the optimal permutation from that Example, that is, 3 mM totalmagnesium and 0.1 U/μL final DNA polymerase at the various steps asdetailed in Example 48. In this Example however, the template DNAsources will be the various patient samples as described earlier in thisExample. In some cases, the isolated patient DNA will be naturallyfragmented, in which case it will not be further fragmented if of theproper size (e.g. approximate average 100 to 500 bp) (e.g. freelycirculating DNA in blood or urine, not arising from blood cells orbladder cells respectively, but from arising non-local sources). Inother cases, isolated patient DNA will not be naturally fragmented (e.g.from properly preserved tissue samples), in which case the DNA will befragmented by direct probe sonication as in Example 48 (e.g. toapproximate average 100 to 500 bp), or via mechanical shearing,enzymatic digestion or nebulization for example, prior to use insolid-phase bridge PCR.

Critically, using the criteria developed in previous Examples, thetemplate DNA will be added to the beads in specific amounts so as toachieve effective amplification of one or a few initially added templatemolecules per bead.

Another important difference from Example 48 is that all of thedifferent Primer-Conjugated Agarose beads sets described earlier in thisExample will be combined into one solid-phase bridge PCR reaction, so asto allow multiplexed amplification of the different primer-targetedsegments of the APC gene, using the same template mixture.

The template amount, reaction volume and numbers of beads will be scaledaccordingly from that used in Example 48, so as to allow detection downto at least 1 mutant APC molecule out of 1,000 total (i.e. 999 wild-typemolecules) for each bead set (each gene segment), with a 5-fold beadredundancy for each bead set. For example, this would entail 5,000 beadsof each bead set and 60,000 beads total per reaction for all 12 APCsegments, corresponding to approximately 60 μL of actual bead volume(see Example 48 for number of beads per μL).

Second Round of Solid-Phase Bridge PCR:

Will be performed as in Example 48, except that following solid-phasebridge PCR, the beads will not be hybridized with an oligonucleotideprobe, but will instead be subjected to antibody coating and multiplexedcell-free protein expression, using in situ capture, as described seebelow.

Attaching the PC-Antibody to Beads Following Solid-Phase Bridge PCR:

The photocleavable binding/capture anti-HSV antibody (the PC-antibody)will be attached to the post solid-phase bridge PCR beads as performedin Example 31.

Multiplexed Cell-Free Protein Expression with In Situ Capture:

Will be performed as in Example 31 using the rabbit reticulocytecell-free expression system (TNT® T7 Quick for PCR DNA; Promega,Madison, Wis.) or as in Example 40, using the PureSystem cell-freeexpression mixture (mixture prepared according to the manufacturer'sinstructions; Post Genome Institute Co., LTD., Japan).

Contact Photo-Transfer and Antibody Probing:

Following protein synthesis with in situ capture, the entire beadpopulation will be photo-printed onto a microarray substrate (contactphoto-transfer) and the printed substrate simultaneously probed with ananti-VSV Cy3 labeled N-terminal detection antibody and an anti-p53 Cy5labeled C-terminal detection antibody as done in Example 42. The probingis performed to detect mutant truncated proteins missing the N-terminalas described in Example 42. Fluorescence imaging is performed as inExample 42. Note that contact photo-transfer can be omitted and theantibody probing performed directly on the beads (e.g. similar toExample 41).

Results:

Beads which originally amplify single APC template moleculescorresponding to a particular APC segment having a truncating mutation,are expected to ultimately carry the truncated protein product followingmultiplexed cell-free protein expression with in situ capture. Hence,following antibody probing for the N-terminal and C-terminal epitopetags, these beads (or bead-derived microarray spots) will be detected aslacking a C-terminus on the expressed protein, as determined by thepresence of the N-terminal antibody probe signal but absence of theC-terminal antibody probe signal. Conversely, beads or bead-derivedmicroarray spots with wild-type APC proteins will have both N-terminaland C-terminal antibody probe signals. The ratio of mutant to wild-typebeads or bead-derived microarray spots is expected to mirror the ratioof mutant and wild-type APC molecules present in the patient sample.

Instead of antibody probing of the protein containing beads orbead-derived spots, other protein-based analyses are possible, such asmass spectrometric analysis, which would detect missense mutations inaddition to truncation (nonsense) mutations, based on a precise massshift of the protein/peptide.

Lastly, protein expression of the beads can be omitted, in favor of DNAlevel assays on the post solid-phase bridge PCR beads. For example,single-base extension or massively parallel DNA sequencing could beemployed for mutation detection on the beads.

Overall, the methodology is expected to allow non-invasive earlydiagnosis of colorectal cancer at the molecular level, with highsensitivity and high throughput screening abilities. The effectivesingle-molecule amplification per bead will facilitate detection of lowabundance mutant DNA molecules (relative to wild-type) in various typesof patient samples. The full multiplexing of the solid-phase bridge PCRand cell-free protein expression (with in situ capture) will allowsimultaneous analysis of different segments of a gene or template aswell as different genes or markers for example.

Example 52 Solid-Phase Bridge PCR Followed by Cell-Free Expression withIn Situ Protein Capture on PC-Antibodies: Background Reduction in MassSpectrometry Analysis by Subsequent Photo-Release Preparing theSolid-Phase Bridge PCR Template DNA:

K562 cell-line total RNA was purchased from the ATCC (Manassas, Va.) andsubjected to RT-PCR using the Advantage RT-PCR Kit (Clontech, MountainView, Calif.) according to the manufacturer's instructions. The secondstep reaction of the RT-PCR was directed against the BCR-ABL transcriptexpressed in K562 cells with the following primers, which result in anapproximate 1.8 kb product:

BCR-ABL RT-PCR Forward: 5′gCgAACAAgggCAgCAAggCTACg3′ [SEQ NO. 99]BCR-ABL RT-PCR Reverse: 5′ACTggATTCCTggAACATTgTTTCAAAggCTTg3′ [SEQ NO.100]The resultant ˜1.8 kb product was used directly as template in thesolid-phase bridge PCR reaction without purification.

Preparation of Agarose Beads Covalently Conjugated to PCR Primers Usedfor Solid-Phase Bridge PCR:

Primer-Conjugated Agarose Beads prepared as in Example 36, except thatthe concentration of each primer during conjugation to the beads was62.5 μM in 100 mM sodium bicarbonate, 1M NaCl as the Binding Buffer. Thefollowing primer pair was used for conjugation to the beads:

Forward: [SEQ NO. 101]5′[Amine]TAATACgACTCACTATAgggAgAggAggTATATCAATggATTATAAAgACgATgATgATAAAAACTACgACAAgTgggAgATg3′ Reverse: [SEQ NO. 102]5′[Amine]TTATTTATTTATCACCgTCAggCTgTATTTCTT3′

The above primers amplify a region of the BCR-ABL tyrosine kinase domaindesignated in this Example as Segment 1. The primers also incorporate anN-terminal FLAG epitope tag for purification of the expressed peptide.

Qualitative Analysis of Primer Attachment:

Performed as in Example 36.

Solid-Phase Bridge PCR:

5 μL actual total bead volume of the previously preparedPrimer-Conjugated Agarose Beads was used per each sample. Beads wereinitially washed using 0.45 micron pore size, PVDF membrane,micro-centrifuge Filtration Devices (Ultrafree-MC DuraporeMicro-centrifuge Filtration Devices, 400 μL capacity; Millipore,Billerica, Mass.). Unless otherwise noted, all washes involving theFiltration Devices were by brief vortex mixing (5 sec), spinning downbriefly in a micro-centrifuge (just until reaches maximum speed of˜3,000 rpm corresponding to ˜16,000×g) and discarding the filtrate.Initial washes were 2×400 μL with TE-50 mM NaCl (10 mM Tris, pH 8.0, 1mM EDTA, 50 mM NaCl). Beads were then resuspended in TE-50 mM NaCl to20% (v/v) and the suspensions recovered into 0.5 mL thin-walledpolypropylene PCR tubes. The tubes were placed in a PCR machine at 95°C. for 10 min to allow heat-mediated washing (lid temperature 105° C.and no mineral oil used) (beads were resuspended by brief gentle vortexmixing just before this step). After heating, the tubes were immediatelyremoved from the PCR machine, the beads were diluted to 400 μL withTE-50 mM NaCl and the bead suspensions were then transferred toFiltration Devices. Filtration was performed and the filtrate discarded.Beads were briefly washed 1×400 μL more with TE-50 mM NaCl then 1×400 μLwith MBG-Water.

Following the final filtration step (wash) on the bead samples, thewashed bead pellets were resuspended in a commercially availablepre-mixed PCR reaction solution (Platinum® PCR SuperMix High Fidelity;contains 22 U/mL complexed recombinant Taq DNA polymerase, Pyrococcusspecies GB-D thermostable polymerase, PlatinumE) Taq Antibody, 66 mMTris-SO₄ pH 8.9, 19.8 mM (NH₄)₂SO₄, 2.4 mM MgSO₄, 220 μM dNTPs andstabilizers; Invitrogen Corporation, Carlsbad, Calif.; solution usedwithout prior dilution), which was supplemented with 0.2 U/μL additionalDNA polymerase (same polymerase as in aforementioned PCR mix) and ˜10 ngof the aforementioned RT-PCR product as template. 10 μL of this mixturewas used per each 1 μL actual bead volume. No soluble primers were used.The suspensions (˜50 μL) were placed into 0.5 mL polypropylene thin-wallPCR tubes. The samples were subjected to the following thermocycling ina PCR machine (lid temperature 105° C. and no mineral oil used): Aninitial denaturing step (once) of 94° C. for 2 min (beads were brieflyresuspended by gentle vortex mixing just before this step), and 40cycles of 94° C. for 30 sec (denature), 65° C. for 30 sec (anneal) and68° C. for 30 sec (extend); followed by a final extension step of 68° C.for 10 min.

400 μL of TE-50 mM NaCl (10 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl) wasadded to each completed solid-phase bridge PCR reaction and thesuspensions transferred to Filtration Devices (Ultrafree-MC DuraporeMicro-centrifuge Filtration Devices, 400 μL capacity; Millipore,Billerica, Mass.). Filtration was performed in a micro-centrifuge (justuntil reaches maximum speed of ˜13,000 rpm corresponding to ˜16,000×g)and the filtrate discarded. The beads were washed 3×400 μL more withTE-50 mM NaCl; resuspending by ˜5 sec vortex mixing then performingfiltration and discarding the filtrate as described earlier in thisExample. At this point beads could either be placed in SP-PCR StorageBuffer (50% glycerol, TE-50 mM NaCl) and stored at −20° C. (5% beadsv/v) or processed immediately as detailed below in this Example.

Attaching the PC-Antibody to Beads Following Solid-Phase Bridge PCR:

Following the solid-phase bridge PCR reaction, the 5 μL actual beadvolume per sample was washed briefly 3×400 μL with TE-Saline (10 mMTris-HCl, pH 8.0, 1 mM EDTA, 200 mM NaCl). Unless otherwise noted, allwashes and bead manipulations were performed in batch mode using 0.45micron pore size, PVDF membrane, micro-centrifuge Filtration Devices tofacilitate manipulation of the beaded matrix (˜100 micron beads) andexchange the buffers (Ultrafree-MC Durapore Micro-centrifuge FiltrationDevices, 400 μL capacity; Millipore, Billerica, Mass.). NeutrAvidin(tetrameric) was then attached to the bead bound biotin-amine linker, inexcess, by treatment with 400 μL of a 0.2 μg/μL solution in TE-Salinefor 20 min (note: biotin-amine linker attached during previouspreparation of Primer-Conjugated Agarose Beads; see Example 36). Beadswere washed briefly 4×400 μL with TE-Saline.

The beads were next coated with a monoclonal mouse anti-FLAG tag captureantibody which was converted to photocleavable form by conjugation toPC-biotin. Creation of the photocleavable antibody (PC-antibody) wasperformed similar to as described in Example 2. To first create thePC-antibody (prepared in advance), 1 mg of antibody as supplied by themanufacturer (Mouse Anti-FLAG M2 Antibody; Sigma-Aldrich, St. Louis,Mo.) was purified on a NAP-5 desalting column according to themanufacturer's instructions (Amersham Biosciences Corp., Piscataway,N.J.) against a 200 mM sodium bicarbonate and 200 mM NaCl buffer(nuclease-free reagents). The resultant antibody was then reacted with25 molar equivalents of AmberGen's PC-biotin-NHS labeling reagent (addedfrom a 50 mM stock in anhydrous DMF) for 30-60 min with gentle mixing.The labeled antibody was then purified on a NAP-10 desalting columnaccording to the manufacturer's instructions (Amersham BiosciencesCorp., Piscataway, N.J.) against TE-Saline buffer. This preparedmonoclonal anti-FLAG PC-biotin conjugate was then loaded onto the beadsby treatment of the beads (still in Filtration Devices) with 200 μL of0.15 μg/μL in TE-Saline for 20 min. Beads were washed briefly 4×400 μLin TE-Saline followed by 2×400 μL in Molecular Biology Grade Water(MBG-Water).

Cell-Free Protein Expression of the Beads and In Situ Protein Capture:

Still in the Filtration Devices, the 5 μL bead pellets were thenresuspended in 50 μL of the E. coli based PureSystem cell-free proteinexpression mixture (mixture prepared according to the manufacturer'sinstructions; Post Genome Institute Co., LTD., Japan) (no soluble DNAwas included in the reaction). The expression mixture was additionallysupplemented with 250 mM final Betaine concentration from a 5M stock(Sigma-Aldrich, St. Louis, Mo.) to minimize mRNA secondary structure.Protein expression was carried out for 1-2 hr at 42° C. in with gentlemixing (in the upper chamber of the Filtration Devices). Afterexpression, filtration was performed and the filtrate discarded. Stillin the Filtration Devices, the beads were washed 1×400 μL with PBS-T[standard PBS with 0.2% Triton X-100 (v/v)]. Beads were then washed2×400 μL with mass spectrometry grade water (MSG-Water). Beads werere-suspended 50 μL of MSG-Water and recovered from the FiltrationDevices.

Elution of Bead Bound Peptides and Mass Spectrometry Analysis:

The aforementioned 50 μL bead suspension was split into equal portionsof 25 μL with each portion going into separate micro-columns.Micro-columns consist of 10 μL volume polypropylene pipette tips crimpedat the end in order to trap the beads (i.e. prevent beads from flowingout of column). Essentially all of the MSG-Water was drained from thecolumn by gravity (although agarose beads remain partially hydrated).One micro-column was eluted by denaturation of the capture antibodywhile the other was photo-eluted (photo-release of the photocleavablecapture antibody). For denaturing elution, 5 μL of MALDI-TOF massspectrometry matrix solution (20 mg/mL sinapinic acid matrix in 50%acetonitrile and 0.1% trifluoroacetic acid) was applied to themicro-column and the first ˜1 μL eluted droplet collected directly ontoa stainless steel MALDI-TOF plate. The droplet was then allowed todry/crystallize under ambient conditions. For photo-elution, the beads,still in the micro-column, were exposed to near-UV light (365 nm peak UVlamp, Blak-Ray Lamp, Model XX-15, UVP, Upland, Calif.) at a 5 cmdistance for 10 minutes. Importantly, the polypropylene micro-columnstransmit the necessary light. The power output under these conditionswas 2.6 mW/cm² at 360 nm, 1.0 mW/cm² at 310 nm and 0.16 mW/cm² at 250nm. Following photo-elution, approximately 5 μL of MSG-Water was appliedto the micro-column and the first ˜1 μL eluted droplet collecteddirectly onto a stainless steel MALDI-TOF plate. The droplet was mixedwith equal volume of the aforementioned MALDI-TOF mass spectrometrymatrix solution and the droplet was then allowed to dry/crystallizeunder ambient conditions. Once dried, the spots were analyzed using aVoyager-DE MALDI-TOF mass spectrometer (Applied Biosystems; Foster City,Calif.).

Results:

Results are shown in FIG. 46. With both the denaturing elution andphoto-elution methods, the correct peptide peak corresponding to theso-called Segment 1 of the BCR-ABL tyrosine kinase domain was identifiedwith a mass accuracy of ±1 Dalton (0.02% mass error). However, in thedenaturing elution method, several contaminating background peaks areobserved which are not present in the photo-eluted sample. Background isbelieved to be caused by 2 mechanisms: First, the highly charged DNA and(strept)avidin on the bead surface, as well as the agarose bead surfaceitself, can mediate non-specific binding of components in the highlyconcentrated cell-free protein expression system. These components canremain bound to the beads even after extensive washing, but are stripedfrom the beads by the denaturing elution, contaminating target peptideand creating background in the mass spectrometry analysis. Second, theDNA and (strept)avidin, present at high concentrations on the beads, canthemselves leach from beads hence directly causing background(especially minor degradation products falling in the mass range ofinterest). The gentle and highly selective photo-elution avoids theseproblems, leaving such contaminants behind on the beads. Lastly, likelybecause of the contaminating materials in the denaturing elution method,the magnitude of the target peak (Segment 1) is 4-fold less than that ofthe photo-eluted peptide.

Example 53 Solid-Phase Bridge PCR Followed by Cell-Free Expression andMass Spectrometry Analysis Multiplex Cell-Free Expression Preparing theSolid-Phase Bridge PCR Template DNA:

K562 cell-line total RNA was purchased from the ATCC (Manassas, Va.) andsubjected to RT-PCR using the Advantage RT-PCR Kit (Clontech, MountainView, Calif.) according to the manufacturer's instructions. The secondstep reaction of the RT-PCR was directed against the BCR-ABL transcriptexpressed in K562 cells with the following primers, which result in anapproximate 1.8 kb product:

BCR-ABL RT-PCR Forward: 5′gCgAACAAgggCAgCAAggCTACg3′ [SEQ NO. 103]BCR-ABL RT-PCR Reverse: 5′ACTggATTCCTggAACATTgTTTCAAAggCTTg3′ [SEQ NO.104]The resultant ˜1.8 kb product was used directly as template in thesolid-phase bridge PCR reaction without purification.

Preparation of Agarose Beads Covalently Conjugated to PCR Primers Usedfor Solid-Phase Bridge PCR:

Primer-Conjugated Agarose Beads prepared as in Example 36, except thatthe concentration of each primer during conjugation to the beads was62.5 μM in 100 mM sodium bicarbonate, 1M NaCl as the Binding Buffer. Thefollowing primer pairs were used for conjugation to the beads (oneprimer pair per batch of beads):

Forward Segment 1: [SEQ NO. 105]5′[Amine]TAATACgACTCACTATAgggAgAggAggTATATCAATggATTATAAAgACgATgATgATAAAAACTACgACAAgTgggAgATg3′ Reverse Segment 1: [SEQ NO.106] 5′[Amine]TTATTTATTTATCACCgTCAggCTgTATTTCTT3′ Forward Segment 2:[SEQ NO. 107] 5′[Amine]TAATACgACTCACTATAgggAgAggAggTATATCAATggATTATAAAgACgATgATgATAAAgTgTACgAgggCgTgTgg3′ Reverse Segment 2: [SEQ NO.108] 5′[Amine]TTATTTATTTATTTCTTTCAAgAACTCTTCCACCTC3′ Forward Segment 3:[SEQ NO. 109] 5′[Amine]TAATACgACTCACTATAgggAgAggAggTATATCAATggATTATAAAgACgATgATgATAAAgCCgTgAAgACCTTgAAggAg3′ Reverse Segment 3: [SEQ NO.110] 5′[Amine]TTATTTATTTATAAggAgCTgCACCAggTTAgg3′ Forward Segment 4:[SEQ NO. 111] 5′[Amine]TAATACgACTCACTATAgggAgAggAggTATATCAATggATTATAAAgACgATgATgATAAAgTCTgCACCCgggAgCC3′ Reverse Segment 4: [SEQ NO.112] 5′[Amine]TTATTTATTTATCACCACggCgTTCACCT3′ Forward Segment 7: [SEQNO. 113] 5′[Amine]TAATACgACTCACTATAgggAgAggAggTATATCAATggATTATAAAgACgATgATgATAAAAACTgCCTggTAggggAgAAC3′ Reverse Segment 7: [SEQ NO.114] 5′[Amine]TTATTTATTTATAgTCCATTTgATggggAACTTg3′ Forward Segment 10:[SEQ NO. 115] 5′[Amine]TAATACgACTCACTATAgggAgAggAggTATATCAATggATTATAAAgACgATgATgATAAACAgTggAATCCCTCTgACC3′ Reverse Segment 10: [SEQ NO.116] 5′[Amine]TTATTTATTTATgCCTTgTTTCCCCAgCTCCTTTTC3′

The above primers amplify regions of the BCR-ABL tyrosine kinase domaindesignated in this Example as Segments 1, 2, 3, 4, 7, 10. The primersalso incorporate a common N-terminal FLAG epitope tag for purificationof all expressed peptides.

Qualitative Analysis of Primer Attachment:

Performed as in Example 36.

Multiplexed Solid-Phase Bridge PCR:

5 μL actual total bead volume of the previously preparedPrimer-Conjugated Agarose Beads was used per each sample. The 5 μL totalbead volume was a mixture of equal amounts of the different beadspecies, prepared as described earlier in this Example, each beadspecies carrying different primer pairs for the different BCR-ABLsegments. Therefore, the subsequently described procedure corresponds toa single multiplexed solid-phase bridge PCR reaction. Beads wereinitially washed using 0.45 micron pore size, PVDF membrane,micro-centrifuge Filtration Devices (Ultrafree-MC DuraporeMicro-centrifuge Filtration Devices, 400 μL capacity; Millipore,Billerica, Mass.). Unless otherwise noted, all washes involving theFiltration Devices were by brief vortex mixing (˜5 sec), spinning downbriefly in a micro-centrifuge (just until reaches maximum speed of˜13,000 rpm corresponding to ˜16,000×g) and discarding the filtrate.Initial washes were 2×400 μL with TE-50 mM NaCl (10 mM Tris, pH 8.0, 1mM EDTA, 50 mM NaCl). Beads were then resuspended in TE-50 mM NaCl to20% (v/v) and the suspensions recovered into 0.5 mL thin-walledpolypropylene PCR tubes. The tubes were placed in a PCR machine at 95°C. for 10 min to allow heat-mediated washing (lid temperature 105° C.and no mineral oil used) (beads were resuspended by brief gentle vortexmixing just before this step). After heating, the tubes were immediatelyremoved from the PCR machine, the beads were diluted to 400 μL withTE-50 mM NaCl and the bead suspensions were then transferred toFiltration Devices. Filtration was performed and the filtrate discarded.Beads were briefly washed 1×400 L more with TE-50 mM NaCl then 1×400 μLwith MBG-Water.

Following the final filtration step (wash) on the bead samples, thewashed bead pellets were resuspended in a commercially availablepre-mixed PCR reaction solution (Platinum® PCR SuperMix High Fidelity;contains 22 U/mL complexed recombinant Taq DNA polymerase, Pyrococcusspecies GB-D thermostable polymerase, Platinum® Taq Antibody, 66 mMTris-SO₄ pH 8.9, 19.8 mM (NH₄)₂SO₄, 2.4 mM MgSO₄, 220 μM dNTPs andstabilizers; Invitrogen Corporation, Carlsbad, Calif.; solution usedwithout prior dilution), which was supplemented with 0.2 U/μL additionalDNA polymerase (same polymerase as in aforementioned PCR mix) and ˜10 ngof the aforementioned RT-PCR product as template. 10 μL of this mixturewas used per each 1 μL actual bead volume. No soluble primers were used.The suspensions (˜50 μL) were placed into 0.5 mL polypropylene thin-wallPCR tubes. The samples were subjected to the following thermocycling ina PCR machine (lid temperature 105° C. and no mineral oil used): Aninitial denaturing step (once) of 94° C. for 2 min (beads were brieflyresuspended by gentle vortex mixing just before this step), and 40cycles of 94° C. for 30 sec (denature), 65° C. for 30 sec (anneal) and68° C. for 30 sec (extend); followed by a final extension step of 68° C.for 10 min.

400 μL of TE-50 mM NaCl (10 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl) wasadded to each completed solid-phase bridge PCR reaction and thesuspensions transferred to Filtration Devices (Ultrafree-MC DuraporeMicro-centrifuge Filtration Devices, 400 μL capacity; Millipore,Billerica, Mass.). Filtration was performed in a micro-centrifuge (justuntil reaches maximum speed of ˜13,000 rpm corresponding to ˜16,000×g)and the filtrate discarded. The beads were washed 3×400 μL more withTE-50 mM NaCl; resuspending by ˜5 sec vortex mixing then performingfiltration and discarding the filtrate as described earlier in thisExample. At this point beads could either be placed in SP-PCR StorageBuffer (50% glycerol, TE-50 mM NaCl) and stored at −20° C. (5% beadsv/v) or processed immediately as detailed below in this Example.

Multiplexed Cell-Free Protein Expression of the Beads:

Using the aforementioned Filtration Devices, 1 μL total of the postsolid-phase bridge PCR beads was washed 3×400 μL more with MBG-Water andthen resuspended in 15 μL of the E. coli based PureSystem cell-freeprotein expression mixture (mixture prepared according to themanufacturer's instructions; Post Genome Institute Co., LTD., Japan) (nosoluble DNA was included in the reaction). The expression mixture wasadditionally supplemented with 250 mM final Betaine concentration from a5M stock (Sigma-Aldrich, St. Louis, Mo.) to minimize mRNA secondarystructure. The bead suspensions were then recovered from theirFiltration Devices and transferred to 0.5 mL polypropylene PCR tubes.Protein expression was carried out for 1-2 hr at 42° C. in with gentlemixing. Because the post solid-phase bridge PCR beads were a mixture ofbeads corresponding to 6 different BCR-ABL segments, a singlemultiplexed cell-free protein expression reaction was used. Afterexpression, beads were spun down in a micro-centrifuge (just untilreaches maximum speed of ˜13,000 rpm corresponding to ˜16,000×g) and thefluid supernatant containing the cell-free expressed peptide mixture wascollected and combined with 40 μL of PBS-T [standard PBS with 0.2%Triton X-100 (v/v)].

Purification and Elution of Bead Bound Peptides and Mass SpectrometryAnalysis:

Micro-columns were used to affinity purify the cell-free expressedpeptides via their common N-terminal FLAG epitope tag. Micro-columnsconsisted of 10 μL volume polypropylene pipette tips crimped at the endin order to trap the affinity beads (i.e. prevent beads from flowing outof column). Micro-columns were pre-loaded with ˜2 μL mouse anti-FLAGantibody coated agarose affinity beads (EZview™ Red Anti-FLAG® M2Affinity Gel, Sigma-Aldrich, St. Louis, Mo.) and pre-washed in excessPBS-T, allowing the liquid to flow by unit gravity. Next, the crudecell-free expressed peptide mixtures were injected into micro-columnsand allowed to flow through by unit gravity. Micro-columns were thenwashed 2×100 μL in PBS-T followed by 2×100 μL mass spectrometry gradewater (MSG-Water). Essentially all of the MSG-Water was drained from thecolumn by unit gravity (although agarose beads remain partiallyhydrated). To elute the peptides from the affinity beads, 5 μL ofMALDI-TOF mass spectrometry matrix solution (20 mg/mL sinapinic acidmatrix in 50% acetonitrile and 0.1% trifluoroacetic acid) was applied tothe micro-column and the first ˜1 μL eluted droplet collected directlyonto a stainless steel MALDI-TOF plate. The droplet was then allowed todry/crystallize under ambient conditions without disturbance. Oncecompletely dried, the spots were analyzed using a Voyager-DE MALDI-TOFmass spectrometer (Applied Biosystems; Foster City, Calif.).

Results:

Results are shown in FIG. 47. The correct peptide peaks corresponding toall 6 segments (Segments 1, 2, 3, 4, 7, and 10) of the BCR-ABL tyrosinekinase domain were identified with a mass accuracy of 1±1 Dalton (0.01%mass error; average error over all 6 peptides). These data demonstrateproof-of-principal that a single multiplexed solid-phase bridge PCRreaction can then be used to mediate a single multiplexed cell-freeprotein expression reaction, which was then followed peptidepurification and mass spectrometry analysis. Note that the additional+75 Da peak, relative to the peak of Segment 7 (determined bynon-multiplex samples), is putatively identified as a SNP, resulting ina G to E amino acid substitution (+72 Da), which was also identified inpreliminary DNA sequencing experiments.

Example 54 Affinity Purification of Cell-Free Expressed Peptides onto anAgarose Bead Affinity Resin Followed by Mass Spectrometry Detection fromSingle Beads PCR to Create Template DNA for Cell-Free ProteinExpression:

Conventional solution-phase PCR was performed on cell-line genomic DNAusing standard molecular biology practices. The PCR product wasconfirmed by conventional agarose gel electrophoresis and used fortemplate in the cell-free protein expression reactions withoutpurification. The following primer pairs were used to amplify so-calledSegment 7 of the APC mutation cluster region (MCR) of Exon 15:

Forward APC Segment 7: [SEQ NO. 117]5′TAATACgACTCACTATAgggAgAggAggTATATCAATgAAAgATTATAAAgACgATgATgATAAATgTACTTCTgTCAgTTCACTT3′ Reverse APC Segment 7: [SEQ NO.118] 5′TTATTTATTTATCATggTTTgTCCAgggCTATC3′

Cell-Free Protein Expression:

Cell-free protein expression was carried out using 1 μL of theaforementioned soluble PCR product in 10 μL of the E. coli basedPureSystem cell-free protein expression mixture (mixture preparedaccording to the manufacturer's instructions; Post Genome Institute Co.,LTD., Japan). The expression mixtures were additionally supplementedwith 250 mM final Betaine concentration from a 5M stock (Sigma-Aldrich,St. Louis, Mo.) to minimize mRNA secondary structure. Protein expressionwas carried out for 1-2 hr at 42° C. in with gentle mixing.

Purification and Elution of Bead Bound Peptides and Mass SpectrometryAnalysis:

The crude cell-free expression mixtures were then diluted with 50 μL ofAB-T [100 mM ammonium bicarbonate with 0.1% Triton X-100 (v/v)]. Mouseanti-FLAG antibody coated agarose affinity beads were used in batch modeto purify the cell-free expressed peptides (EZview™ Red Anti-FLAG® M2Affinity Gel, Sigma-Aldrich, St. Louis; binding capacity for FLAG-taggedproteins is >100 ng per 1 μl of packed gel). The diluted crude cell-freeexpression mixtures were combined directly with ˜1 μL beads in 0.5 mLpolypropylene PCR tubes. The mixtures were then incubated for 20 minutesat room temperature with gentle mixing to keep the beads suspended. Thebeads were then spun down in a micro-centrifuge (just until reachesmaximum speed of ˜13,000 rpm corresponding to ˜16,000×g) and the fluidsupernatant removed and discarded. The beads were then washed 2×10minutes each in mass spectrometry grade water (MSG-Water), removing thefluid supernatant as before. After removing the final wash, beads wereresuspended in 50 mL MSG-Water and individual beads were selected fromsuspension by careful pipetting and deposited onto a stainless steelMALDI-TOF plate. A small volume (0.2-0.5 μL) of MALDI-TOF matrixsolution (20 mg/mL sinapinic acid matrix in 50% acetonitrile and 0.1%trifluoroacetic acid) was immediately applied directly on top of thebeads. The droplet was then allowed to dry/crystallize under ambientconditions without disturbance. The size of the final spot wasapproximately 2 mm in diameter with the beads near the center of spot.Once completely dried, the spots were analyzed using a Voyager-DEMALDI-TOF mass spectrometer (Applied Biosystems; Foster City, Calif.).The MALDI-TOF spectra were acquired on the outer edge of the spot,inside the spot in the immediate vicinity of the beads and also directlyfrom the beads. The signal intensity was typically higher near thebeads, although the matrix solution can elute peptides from the beadsresulting in peptide spreading prior to drying of the matrix solutionspot.

Results:

FIG. 48 shows that the expected peptide, corresponding to so-calledSegment 7 of the APC gene, was observed at the correct mass (expectedpeptide mass 6,203 Da including N-terminal formylation produced in thecell-free expression system). These data confirm that the amount ofpeptide that can be bound to single agarose beads of roughly 100 micronsin diameter, is sufficient to be detected by MALDI-TOF massspectrometry. This is consistent with the reported capacity of theagarose beads, which at >100 ng/μL beads and approximately 1,000individual beads per μL bead volume, would amount to approximately 20femtomoles of a 6,000 Da peptide. This falls within range of thesensitivity of MALDI-TOF mass spectrometry.

Template Sequences for Experimental

Template: Example 25p53 in pETBlue-2 Plasmid; p53 portion is GeneBankNM_(—)000546[SEQ NO. 119]

5′gCCggCACCTgTCCTACgAgTTgCATgATAAAgAAgACAgTCATAAgTgCggCgACgACCggTgAATTgTgAgCgCTCACAATTCTCgTgACATCATAACgTCCCgCgAAATTAATACgACTCACTATAggggAATTgTgAgCggATAACAATTCCCCTCTAgACTTACAATTTCCATTCgCCATTCAggCTgCgCAACTgTTgggAAgggCgATCggTACgggCCTCTTCgCTATTACgCCAgCTTgCgAACggTgggTgCgCTgCAAggCgATTAAgTTgggTAACgCCAggATTCTCCCAgTCACgACgTTgTAAAACgACggCCAgCgAgAgATCTTgATTggCTAgCAgAATAATTTTgTTTAACTTTAAgAAggAgATATACCATggCgATAgAggAgCCgCAgTCAgATCCTAgCgTCgAgCCCCCTCTgAgTCAggAAACATTTTCAgACCTATggAAACTACTTCCTgAAAACAACgTTCTgTCCCCCTTgCCgTCCCAAgCAATggATgATTTgATgCTgTCCCCggACgATATTgAACAATggTTCACTgAAgACCCAggTCCAgATgAAgCTCCCAgAATgCCAgAggCTgCTCCCCgCgTggCCCCTgCACCAgCAgCTCCTACACCggCggCCCCTgCACCAgCCCCCTCCTggCCCCTgTCATCTTCTgTCCCTTCCCAgAAAACCTACCAgggCAgCTACggTTTCCgTCTgggCTTCTTgCATTCTgggACAgCCAAgTCTgTgACTTgCACgTACTCCCCTgCCCTCAACAAgATgTTTTgCCAACTggCCAAgACCTgCCCTgTgCAgCTgTgggTTgATTCCACACCCCCgCCCggCACCCgCgTCCgCgCCATggCCATCTACAAgCAgTCACAgCACATgACggAggTTgTgAggCgCTgCCCCCACCATgAgCgCTgCTCAgATAgCgATggTCTggCCCCTCCTCAgCATCTTATCCgAgTggAAggAAATTTgCgTgTggAgTATTTggATgACAgAAACACTTTTCgACATAgTgTggTggTgCCCTATgAgCCgCCTgAggTTggCTCTgACTgTACCACCATCCACTACAACTACATgTgTAACAgTTCCTgCATgggCggCATgAACCggAggCCCATCCTCACCATCATCACACTggAAgACTCCAgTggTAATCTACTgggACggAACAgCTTTgAggTgCgTgTTTgTgCCTgTCCTgggAgAgACCggCgCACAgAggAAgAgAATCTCCgCAAgAAAggggAgCCTCACCACgAgCTgCCCCCAgggAgCACTAAgCgAgCACTgCCCAACAACACCAgCTCCTCTCCCCAgCCAAAgAAgAAACCACTggATggAgAATATTTCACCCTTCAgATCCgTgggCgTgAgCgCTTCgAgATgTTCCgAgAgCTgAATgAggCCTTggAACTCAAggATgCCCAggCTgggAAggAgCCAggggggAgCAgggCTCACTCCAgCCACCTgAAgTCCAAAAAgggTCAgTCTACCTCCCgCCATAAAAAACTCATgTTCAAgACAgAAgggCCTgACTCAgACTCCCgggAgCTCgTggATCCgAATTCTgTACAggCgCgCCTgCAggACgTCgACggTACCATCgATACgCgTTCgAAgCTTgCggCCgCACAgCTgTATACACgTgCAAgCCAgCCAgAACTCgCTCCTgAAgACCCAgAggATCTCgAgCACCACCACCACCACCACTAATgTTAATTAAgTTgggCgTTgTAATCATAgTCATAATCAATACTCCTgACTgCgTTAgCAATTTAACTgTgATAAACTACCgCATTAAAgCTATTCgATgATAAgCTgTCAAACATgATAATTCTTgAAgACgAAAgggCCTAggCTgATAAAACAgAATTTgCCTggCggCAgTAgCgCggTggTCCCACCTgACCCCATgCCgAACTCAgAAgTgAAACgCCgTAgCgCCgATggTAgTgTggggTCTCCCCATgCgAgAgTAgggAACTgCCAggCATCAAATAAAACgAAAggCTCAgTCgAAAgACTgggCCTTTCgTTTTATCTgTTgTTTgTCggTgAACgCTCTCCTgAgTAggACAAATCCgCCgggAgCggATTTgAACgTTgCgAAgCAACggCCCggAgggTggCgggCAggACgCCCgCCATAAACTgCCAggCATCAAATTAAgCAgAAggCCATCCTgACggATggCCTTTTTgCgTTTCTACAAACTCTTTTgTTTATTTTTCTAAATACATTCAAATATgTATCCgCTgAgCAATAACTAgCATAACCCCTTggggCCTCTAAACgggTCTTgAggggTTTTTTgCTgAAAggAggAACTATATCCggATTggCgAATgggACgCgCCCTgTAgCggCgCATTAAgCgCggCgggTgTggTggTTACgCgCAgCgTgACCgCTACACTTgCCAgCgCCCTAgCgCCCgCTCCTTTCgCTTTCTTCCCTTCCTTTCTCgCCACgTTgCCggCTTTCCCCgTCAAgCTCTAAATCgggggCTCCCTTTAgggTTCCgATTTAgTgCTTTACggCACCTCgACCCCAAAAAACTTgATTAgggTgATggTTCACgTAgTgggCCATCgCCCTgATAgACggTTTTTCgCCCTTTgACgTTggAgTCCACgTTCTTTAATAgTggACTCTTgTTCCAAACTggAACAACACTCAACCCTATCTCggTCTATTCTTTTgATTTATAAgggATTTTgCCgATTTCggCCTATTggTTAAAAAATgAgCTgATTTAACAAAAATTTAACgCgAATTTTAACAAAATATTAACgTTTACAATTTCTggCggCACgATggCATgAgATTATCAAAAAggATCTTCACCTAgATCCTTTTAAATTAAAAATgAAgTTTTAAATCAATCTAAAgTATATATgAgTAAACTTggTCTgACAgTTACCAATgCTTAATCAgTgAggCACCTATCTCAgCgATCTgTCTATTTCgTTCATCCATAgTTgCCTgACTCCCCgTCgTgTAgATAACTACgATACgggAgggCTTACCATCTggCCCCAgTgCTgCAATgATACCgCgAgACCCACgCTCACCggCTCCAgATTTATCAgCAATAAACCAgCCAgCCggAAgggCCgAgCgCAgAAgTggTCCTgCAACTTTATCCgCCTCCATCCAgTCTATTAATTgTTgCCgggAAgCTAgAgTAAgTAgTTCgCCAgTTAATAgTTTgCgCAACgTTgTTgCCATTgCTACAggCATCgTggTgTCACgCTCgTCgTTTggTATggCTTCATTCAgCTCCggTTCCCAACgATCAAggCgAgTTACATgATCCCCCATgTTgTgCAAAAAAgCggTTAgCTCCTTCggTCCTCCgATCgTTgTCAgAAgTAAgTTggCCgCAgTgTTATCACTCATggTTATggCAgCACTgCATAATTCTCTTACTgTCATgCCATCCgTAAgATgCTTTTCTgTgACTggTgAgTACTCAACCAAgTCATTCTgAgAATAgTgTATgCggCgACCgAgTTgCTCTTgCCCggCgTCAATACgggATAATACCgCgCCACATAgCAgAACTTTAAAAgTgCTCATCATTggAAAACgTTCTTCggggCgAAAACTCTCAAggATCTTACCgCTgTTgAgATCCAgTTCgATgTAACCCACTCgTgCACCCAACTgATCTTCAgCATCTTTTACTTTCACCAgCgTTTCTgggTgAgCAAAAACAggAAggCAAAATgCCgCAAAAAAgggAATAAgggCgACACggAAATgTTgAATACTCATACTCTTCCTTTTTCAATCATgACCAAAATCCCTTAACgTgAgTTTTCgTTCCACTgAgCgTCAgACCCCgTAgAAAAgATCAAAggATCTTCTTgAgATCCTTTTTTTCTgCgCgTAATCTgCTgCTTgCAAACAAAAAAACCACCgCTACCAgCggTggTTTgTTTgCCggATCAAgAgCTACCAACTCTTTTTCCgAAggTAACTggCTTCAgCAgAgCgCAgATACCAAATACTgTCCTTCTAgTgTAgCCgTAgTTAggCCACCACTTCAAgAACTCTgTAgCACCgCCTACATACCTCgCTCTgCTAATCCTgTTACCAgTggCTgCTgCCAgTggCgATAAgTCgTgTCTTACCgggTTggACTCAAgACgATAgTTACCggATAAggCgCAgCggTCgggCTgAACggggggTTCgTgCACACAgCCCAgCTTggAgCgAACgACCTACACCgAACTgAgATACCTACAgCgTgAgCTATgAgAAAgCgCCACgCTTCCCgAAgggAgAAAggCggACAggTATCCggTAAgCggCAgggTCggAACAggAgAgCgCACgAgggAgCTTCCAgggggAAACgCCTggTATCTTTATAgTCCTgTCgggTTTCgCCACCTCTgACTTgAgCgTCgATTTTTgTgATgCTCgTCAggggggCggAgCCTATggAAAAACgCCAgCAACgCggCCTTTTTACggTTCCTggCCTTTTgCTggCCTTTTgCTCACATgTTCTTTCCTgCgTTATCCCCTgATTCTgTggATAACCgTATTACCgCCTTTgAgTgAgCTgATACCgCTCgCCgCAgCCgAACgACCgAgCgCAgCgAgTCAgTgAgCgAggAAgCCggCgATAATggCCTgCTTCTCgCCgAAACgTTTggTggCgggACCAgTgACgAAggCTTgAgCgAgggCgTgCAAgATTCCgAATACCgCAAgCgACAggCCgATCATCgTCgCgCTCCAgCgAAAgCggTCCTCgCCgAAAATgACCCAgAgCgCT3′Template: Example 25 and 30 (Solution PCR Template) GST A2 in pETBlue-2Plasmid; GST A2 portion is GeneBank NM_(—)000846 [SEQ NO. 120]

5′gCCggCACCTgTCCTACgAgTTgCATgATAAAgAAgACAgTCATAAgTgCggCgACgACCggTgAATTgTgAgCgCTCACAATTCTCgTgACATCATAACgTCCCgCgAAATTAATACgACTCACTATAggggAATTgTgAgCggATAACAATTCCCCTCTAgACTTACAATTTCCATTCgCCATTCAggCTgCgCAACTgTTgggAAgggCgATCggTACgggCCTCTTCgCTATTACgCCAgCTTgCgAACggTgggTgCgCTgCAAggCgATTAAgTTgggTAACgCCAggATTCTCCCAgTCACgACgTTgTAAAACgACggCCAgCgAgAgATCTTgATTggCTAgCAgAATATTTTgTTTAACTTTAAgAAggAgATATACCATggCgATAgCAgAgAAgCCCAAgCTCCACTACTCCAATATACggggCAgAATggAgTCCATCCggTggCTCCTggCTgCAgCTggAgTAgAgTTTgAAgAgAAATTTATAAAATCTgCAgAAgATTTggACAAgTTAAgAAATgATggATATTTgATgTTCCAgCAAgTgCCAATggTTgAgATTgATgggATgAAgCTggTgCAgACCAgAgCCATTCTCAACTACATTgCCAgCAAATACAACCTCTATgggAAAgACATAAAggAgAAAgCCCTgATTgATATgTATATAgAAggTATAgCAgATTTgggTgAAATgATCCTTCTTCTgCCCTTTACTCAACCTgAggAACAAgATgCCAAgCTTgCCTTgATCCAAgAgAAAACAAAAAATCgCTACTTCCCTgCCTTTgAAAAAgTCTTAAAgAgCCACggACAAgACTACCTTgTTggCAACAAgCTgAgCCgggCTgACATTCACCTggTggAACTTCTCTACTACgTggAAgAgCTTgACTCTAgCCTTATTTCCAgCTTCCCTCTgCTgAAggCCCTgAAAACCAgAATCAgTAACCTgCCCACAgTgAAgAAgTTTCTACAgCCTggCAgCCCAAggAAgCCTCCCATggATgAgAAATCTTTAgAAgAATCAAggAAgATTTTCAggTTTTCCCgggAgCTCgTggATCCgAATTCTgTACAggCgCgCCTgCAggACgTCgACggTACCATCgATACgCgTTCgAAgCTTgCggCCgCACAgCTgTATACACgTgCAAgCCAgCCAgAACTCgCTCCTgAAgACCCAgAggATCTCgAgCACCACCACCACCACCACTAATgTTAATTAAgTTgggCgTTgTAATCATAgTCATAATCAATACTCCTgACTgCgTTAgCAATTTAACTgTgATAAACTACCgCATTAAAgCTATTCgATgATAAgCTgTCAAACATgATAATTCTTgAAgACgAAAgggCCTAggCTgATAAAACAgAATTTgCCTggCggCAgTAgCgCggTggTCCCACCTgACCCCATgCCgAACTCAgAAgTgAAACgCCgTAgCgCCgATggTAgTgTggggTCTCCCCATgCgAgAgTAgggAACTgCCAggCATCAAATAAAACgAAAggCTCAgTCgAAAgACTgggCCTTTCgTTTTATCTgTTgTTTgTCggTgAACgCTCTCCTgAgTAggACAAATCCgCCgggAgCggATTTgAACgTTgCgAAgCAACggCCCggAgggTggCgggCAggACgCCCgCCATAAACTgCCAggCATCAAATTAAgCAgAAggCCATCCTgACggATggCCTTTTTgCgTTTCTACAAACTCTTTTgTTTATTTTTCTAAATACATTCAAATATgTATCCgCTgAgCAATAACTAgCATAACCCCTTggggCCTCTAAACgggTCTTgAggggTTTTTTgCTgAAAggAggAACTATATCCggATTggCgAATgggACgCgCCCTgTAgCggCgCATTAAgCgCggCgggTgTggTggTTACgCgCAgCgTgACCgCTACACTTgCCAgCgCCCTAgCgCCCgCTCCTTTCgCTTTCTTCCCTTCCTTTCTCgCCACgTTCgCCggCTTTCCCCgTCAAgCTCTAAATCgggggCTCCCTTTAgggTTCCgATTTAgTgCTTTACggCACCTCgACCCCAAAAAACTTgATTAgggTgATggTTCACgTAgTgggCCATCgCCCTgATAgACggTTTTTCgCCCTTTgACgTTggAgTCCACgTTCTTTAATAgTggACTCTTgTTCCAAACTggAACAACACTCAACCCTATCTCggTCTATTCTTTTgATTTATAAgggATTTTgCCgATTTCggCCTATTggTTAAAAAATgAgCTgATTTAACAAAAATTTAACgCgAATTTTAACAAAATATTAACgTTTACAATTTCTggCggCACgATggCATgAgATTATCAAAAAggATCTTCACCTAgATCCTTTTAAATTAAAAATgAAgTTTTAAATCAATCTAAAgTATATATgAgTAAACTTggTCTgACAgTTACCAATgCTTAATCAgTgAggCACCTATCTCAgCgATCTgTCTATTTCgTTCATCCATAgTTgCCTgACTCCCCgTCgTgTAgATAACTACgATACgggAgggCTTACCATCTggCCCCAgTgCTgCAATgATACCgCgAgACCCACgCTCACCggCTCCAgATTTATCAgCAATAAACCAgCCAgCCggAAgggCCgAgCgCAgAAgTggTCCTgCAACTTTATCCgCCTCCATCCAgTCTATTAATTgTTgCCgggAAgCTAgAgTAAgTAgTTCgCCAgTTAATAgTTTgCgCAACgTTgTTgCCATTgCTACAggCATCgTggTgTCACgCTCgTCgTTTggTATggCTTCATTCAgCTCCggTTCCCAACgATCAAggCgAgTTACATgATCCCCCATgTTgTgCAAAAAAgCggTTAgCTCCTTCggTCCTCCgATCgTTgTCAgAAgTAAgTTggCCgCAgTgTTATCACTCATggTTATggCAgCACTgCATAATTCTCTTACTgTCATgCCATCCgTAAgATgCTTTTCTgTgACTggTgAgTACTCAACCAAgTCATTCTgAgAATAgTgTATgCggCgACCgAgTTgCTCTTgCCCggCgTCAATACgggATAATACCgCgCCACATAgCAgAACTTTAAAAgTgCTCATCATTggAAAACgTTCTTCggggCgAAAACTCTCAAggATCTTACCgCTgTTgAgATCCAgTTCgATgTAACCCACTCgTgCACCCAACTgATCTTCAgCATCTTTTACTTTCACCAgCgTTTCTgggTgAgCAAAAACAggAAggCAAAATgCCgCAAAAAAgggAATAAgggCgACACggAAATgTTgAATACTCATACTCTTCCTTTTTCAATCATgACCAAAATCCCTTAACgTgAgTTTTCgTTCCACTgAgCgTCAgACCCCgTAgAAAAgATCAAAggATCTTCTTgAgATCCTTTTTTTCTgCgCgTAATCTgCTgCTTgCAAACAAAAAAACCACCgCTACCAgCggTggTTTgTTTgCCggATCAAgAgCTACCAACTCTTTTTCCgAAggTAACTggCTTCAgCAgAgCgCAgATACCAAATACTgTCCTTCTAgTgTAgCCgTAgTTAggCCACCACTTCAAgAACTCTgTAgCACCgCCTACATACCTCgCTCTgCTAATCCTgTTACCAgTggCTgCTgCCAgTggCgATAAgTCgTgTCTTACCgggTTggACTCAAgACgATAgTTACCggATAAggCgCAgCggTCgggCTgAACggggggTTCgTgCACACAgCCCAgCTTggAgCgAACgACCTACACCgAACTgAgATACCTACAgCgTgAgCTATgAgAAAgCgCCACgCTTCCCgAAgggAgAAAggCggACAggTATCCggTAAgCggCAgggTCggAACAggAgAgCgCACgAgggAgCTTCCAgggggAAACgCCTggTATCTTTATAgTCCTgTCgggTTTCgCCACCTCTgACTTgAgCgTCgATTTTTgTgATgCTCgTCAggggggCggAgCCTATggAAAAACgCCAgCAACgCggCCTTTTTACggTTCCTggCCTTTTgCTggCCTTTTgCTCACATgTTCTTTCCTgCgTTATCCCCTgATTCTgTggATAACCgTATTACCgCCTTTgAgTgAgCTgATACCgCTCgCCgCAgCCgAACgACCgAgCgCAgCgAgTCAgTgAgCgAggAAgCCggCgATAATggCCTgCTTCTCgCCgAAACgTTTggTggCgggACCAgTgACgAAggCTTgAgCgAgggCgTgCAAgATTCCgAATACCgCAAgCgACAggCCgATCATCgTCgCgCTCCAgCgAAAgCggTCCTCgCCgA AAATgACCCAgAgCgCT3′Template: Example 30 GST A2 Solid-Phase Bridge PCR Template; Template islinear construct derived from GST A2 in pETBlue-2 plasmid; GST A2portion is GeneBank NM 000846 [SEQ NO. 121]

5′TgAgCgCTCACAATTCTCgTgACATCATAACgTCCCgCgAAATTAATACgACTCACTATAggggAATTgTgAgCggATAACAATTCCCCTCTAgACTTACAATTTCCATTCgCCATTCAggCTgCgCAACTgTTgggAAgggCgATCggTACgggCCTCTTCgCTATTACgCCAgCTTgCgAACggTgggTgCgCTgCAAggCgATTAAgTTgggTAACgCCAggATTCTCCCAgTCACgACgTTgTAAAACgACggCCAgCgAgAgATCTTgATTggCTAgCAgAATAATTTTgTTTAACTTTAAgAAggAgATATACCATggCgATAgCAgAgAAgCCCAAgCTCCACTACTCCAATATACggggCAgAATggAgTCCATCCggTggCTCCTggCTgCAgCTggAgTAgAgTTTgAAgAgAAATTTATAAAATCTgCAgAAgATTTggACAAgTTAAgAAATgATggATATTTgATgTTCCAgCAAgTgCCAATggTTgAgATTgATgggATgAAgCTggTgCAgACCAgAgCCATTCTCAACTACATTgCCAgCAAATACAACCTCTATgggAAAgACATAAAggAgAAAgCCCTgATTgATATgTATATAgAAggTATAgCAgATTTgggTgAAATgATCCTTCTTCTgCCCTTTACTCAACCTgAggAACAAgATgCCAAgCTTgCCTTgATCCAAgAgAAAACAAAAAATCgCTACTTCCCTgCCTTTgAAAAAgTCTTAAAgAgCCACggACAAgACTACCTTgTTggCAACAAgCTgAgCCgggCTgACATTCACCTggTggAACTTCTCTACTACgTggAAgAgCTTgACTCTAgCCTTATTTCCAgCTTCCCTCTgCTgAAggCCCTgAAAACCAgAATCAgTAACCTgCCCACAgTgAAgAAgTTTCTACAgCCTggCAgCCCAAggAAgCCTCCCATggATgAgAAATCTTTAgAAgAATCAAggAAgATTTTCAggTTTTCCCgggAgCTCgTggATCCgAATTCTgTACAggCgCgCCTgCAggACgTCgACggTACCATCgATACgCgTTCgAAgCTTgCggCCgCACAgCTgTATACACgTgCAAgCCAgCCAgAACTCgCTCCTgAAgACCCAgAggATCTCgAgCACCACCACCACCACCACTAATgTTAATTAAgTTgggCgTTgTAATCATAgTCATAATCAATACTCCTgACTgCgTTAgCAATTTAACTgTgATAAACTACCgCAT TAAAgCTATTCg3′Template: Example 28 & 44 APC Segment 3 of Exon 15; GeneBank of full APCcoding sequence is M74088; Example 54 APC Segment 7 also within belowsequence [bracketed region] [SEQ NO. 122]

5′gTTTCTCCATACAggTCACggggAgCCAATggTTCAgAAACAAATCgAgTgggTTCTAATCATggAATTAATCAAAATgTAAgCCAgTCTTTgTgTCAAgAAgATgACTATgAAgATgATAAgCCTACCAATTATAgTgAACgTTACTCTgAAgAAgAACAgCATgAAgAAgAAgAgAgACCAACAAATTATAgCATAAAATATAATgAAgAgAAACgTCATgTggATCAgCCTATTgATTATAgTTTAAAATATgCCACAgATATTCCTTCATCACAgAAACAgTCATTTTCATTCTCAAAgAgTTCATCTggACAAAgCAgTAAAACCgAACATATgTCTTCAAgCAgTgAgAATACgTCCACACCTTCATCTAATgCCAAgAggCAgAATCAgCTCCATCCAAgTTCTgCACAgAgTAgAAgTggTCAgCCTCAAAAggCTgCCACTTgCAAAgTTTCTTCTATTAACCAAgAAACAATACAgACTTATTgTgTAgAAgATACTCCAATATgTTTTTCAAgATgTAgTTCATTATCATCTTTgTCATCAgCTgAAgATgAAATAggATgTAATCAgACgACACAggAAgCAgATTCTgCTAATACCCTgCAAATAgCAgAAATAAAAgAAAAgATTggAACTAggTCAgCTgAAgATCCTgTgAgCgAAgTTCCAgCAgTgTCACAgCACCCTAgAACCAAATCCAgCAgACTgCAgggTTCTAgTTTATCTTCAgAATCAgCCAggCACAAAgCTgTTgAATTTTCTTCAggAgCgAAATCTCCCTCCAAAAgTggTgCTCAgACACCCAAAAgTCCACCTgAACACTATgTTCAggAgACCCCACTCATgTTTAgCAgA[TgTACTTCTgTCAgTTCACTTgATAgTTTTgAgAgTCgTTCgATTgCCAgCTCCgTTCAgAgTgAACCATgCAgTggAATggTAAgTggCATTATAAgCCCCAgTgATCTTCCAgATAgCCCTggACAAACCATg]CCACCAAgCAgAAgTAAAACACCTCCACCACCTCCTCAAACAgCTCAAACCAAgCgAgAAgTACCTAAAAATAAAgCACCTACTgCTgAAAAgAgAgAgAgTggACCTAAgCAAgCTgCAgTAAATgCTgCAgTTCAgAgggTCCAggTTCTTCCAgATgCTgATACTTTATTACATTTTgCCACggAAAgTACTCCAgATggATTTTCTTgTTCATCCAgCCTgAgTgCTCTgAgCCTCgATgAgCCATTTATACAgAAAgATgTggAATTAAgAATAATgCCTCCAgTTCAggAAAATgACAATgggAATgAAACAgAATCAgAgCAgCCTAAAgAATCAAATgAAAACCAAgAgAAAgAggCAgAAAAAACTATTgATTCTgAAAAggACCTATTAgATgATTCAgATgATgATgATATTgAAATACTAgAAgAATgTATTATTTCTgCCATgCCAACAAAgTCATCACgTAAAgCAAAAAAgCCAgCCCAgACTgCTTCAAAATTACCTCCACCTgTggCAAggAAACCAAgTCAgCTgCCTgTgTACAAACTTCTACCATCACAAAACAggTTgCAACCCCAAAAgCATgTTAgTTTTACACCgggggATgATATgCCACgggTgTATTgTgTTgAAgggACACCTATAAACTTTTCCACAgCTACATCTCTAAgTgATCTAACAATCgAATCCCCTCCAAATgAgTTAgCTgCTggAgAAggAgTTAgAggAggAgCACAgTCAggTgAATTTgAAAAACgAgATACCATTCCTACAgAAggCAgAAgT3′Template: Example 31 Gamma-Actin GeneBank NM_(—)001614; Full codingsequence [SEQ NO. 123]

5′ATggAAgAAgAgATCgCCgCgCTggTCATTgACAATggCTCCggCATgTgCAAAgCTggTTTTgCTggggACgACgCTCCCCgAgCCgTgTTTCCTTCCATCgTCgggCgCCCCAgACACCAgggCgTCATggTgggCATgggCCAgAAggACTCCTACgTgggCgACgAggCCCAgAgCAAgCgTggCATCCTgACCCTgAAgTACCCCATTgAgCATggCATCgTCACCAACTgggACgACATggAgAAgATCTggCACCACACCTTCTACAACgAgCTgCgCgTggCCCCggAggAgCACCCAgTgCTgCTgACCgAggCCCCCCTgAACCCCAAggCCAACAgAgAgAAgATgACTCAgATTATgTTTgAgACCTTCAACACCCCggCCATgTACgTggCCATCCAggCCgTgCTgTCCCTCTACgCCTCTgggCgCACCACTggCATTgTCATggACTCTggAgACggggTCACCCACACggTgCCCATCTACgAgggCTACgCCCTCCCCCACgCCATCCTgCgTCTggACCTggCTggCCgggACCTgACCgACTACCTCATgAAgATCCTCACTgAgCgAggCTACAgCTTCACCACCACggCCgAgCgggAAATCgTgCgCgACATCAAggAgAAgCTgTgCTACgTCgCCCTggACTTCgAgCAggAgATggCCACCgCCgCATCCTCCTCTTCTCTggAgAAgAgCTACgAgCTgCCCgATggCCAggTCATCACCATTggCAATgAgCggTTCCggTgTCCggAggCgCTgTTCCAgCCTTCCTTCCTgggTATggAATCTTgCggCATCCACgAgACCACCTTCAACTCCATCATgAAgTgTgACgTggACATCCgCAAAgACCTgTACgCCAACACggTgCTgTCgggCggCACCACCATgTACCCgggCATTgCCgACAggATgCAgAAggAgATCACCgCCCTggCgCCCAgCACCATgAAgATCAAgATCATCgCACCCCCAgAgCgCAAgTACTCggTgTggATCggTggCTCCATCCTggCCTCACTgTCCACCTTCCAgCAgATgTggATTAgCAAgCAggAgTACgACgAgTCgggCCCCTCCATCgTCCACCgCAAATgCTTCTAA3′Template: Example 31p53 GeneBank NM_(—)000546; Full coding sequence [SEQNO. 124]

5′ATggAggAgCCgCAgTCAgATCCTAgCgTCgAgCCCCCTCTgAgTCAggAAACATTTTCAgACCTATggAAACTACTTCCTgAAAACAACgTTCTgTCCCCCTTgCCgTCCCAAgCAATggATgATTTgATgCTgTCCCCggACgATATTgAACAATggTTCACTgAAgACCCAggTCCAgATgAAgCTCCCAgAATgCCAgAggCTgCTCCCCgCgTggCCCCTgCACCAgCAgCTCCTACACCggCggCCCCTgCACCAgCCCCCTCCTggCCCCTgTCATCTTCTgTCCCTTCCCAgAAAACCTACCAgggCAgCTACggTTTCCgTCTgggCTTCTTgCATTCTgggACAgCCAAgTCTgTgACTTgCACgTACTCCCCTgCCCTCAACAAgATgTTTTgCCAACTggCCAAgACCTgCCCTgTgCAgCTgTgggTTgATTCCACACCCCCgCCCggCACCCgCgTCCgCgCCATggCCATCTACAAgCAgTCACAgCACATgACggAggTTgTgAggCgCTgCCCCCACCATgAgCgCTgCTCAgATAgCgATggTCTggCCCCTCCTCAgCATCTTATCCgAgTggAAggAAATTTgCgTgTggAgTATTTggATgACAgAAACACTTTTCgACATAgTgTggTggTgCCCTATgAgCCgCCTgAggTTggCTCTgACTgTACCACCATCCACTACAACTACATgTgTAACAgTTCCTgCATgggCggCATgAACCggAggCCCATCCTCACCATCATCACACTggAAgACTCCAgTggTAATCTACTgggACggAACAgCTTTgAggTgCgTgTTTgTgCCTgTCCTgggAgAgACCggCgCACAgAggAAgAgAATCTCCgCAAgAAAggggAgCCTCACCACgAgCTgCCCCCAgggAgCACTAAgCgAgCACTgCCCAACAACACCAgCTCCTCTCCCCAgCCAAAgAAgAAACCACTggATggAgAATATTTCACCCTTCAgATCCgTgggCgTgAgCgCTTCgAgATgTTCCgAgAgCTgAATgAggCCTTggAACTCAAggATgCCCAggCTgggAAggAgCCAggggggAgCAgggCTCACTCCAgCCACCTgAAgTCCAAAAAgggTCAgTCTACCTCCCgCCATAAAAAACTCATgTTCAAgACAgAAgggCCTgACTCAgACTgA3′Template: Example 34 BRCA2 GeneBank NM_(—)000059, exon 11; Full codingsequence [SEQ NO. 125]

ATgCCTATTggATCCAAAgAgAggCCAACATTTTTTgAAATTTTTAAgACACgCTgCAACAAAgCAgATTTAggACCAATAAgTCTTAATTggTTTgAAgAACTTTCTTCAgAAgCTCCACCCTATAATTCTgAACCTgCAgAAgAATCTgAACATAAAAACAACAATTACgAACCAAACCTATTTAAAACTCCACAAAggAAACCATCTTATAATCAgCTggCTTCAACTCCAATAATATTCAAAgAgCAAgggCTgACTCTgCCgCTgTACCAATCTCCTgTAAAAgAATTAgATAAATTCAAATTAgACTTAggAAggAATgTTCCCAATAgTAgACATAAAAgTCTTCgCACAgTgAAAACTAAAATggATCAAgCAgATgATgTTTCCTgTCCACTTCTAAATTCTTgTCTTAgTgAAAgTCCTgTTgTTCTACAATgTACACATgTAACACCACAAAgAgATAAgTCAgTggTATgTgggAgTTTgTTTCATACACCAAAgTTTgTgAAgggTCgTCAgACACCAAAACATATTTCTgAAAgTCTAggAgCTgAggTggATCCTgATATgTCTTggTCAAgTTCTTTAgCTACACCACCCACCCTTAgTTCTACTgTgCTCATAgTCAgAAATgAAgAAgCATCTgAAACTgTATTTCCTCATgATACTACTgCTAATgTgAAAAgCTATTTTTCCAATCATgATgAAAgTCTgAAgAAAAATgATAgATTTATCgCTTCTgTgACAgACAgTgAAAACACAAATCAAAgAgAAgCTgCAAgTCATggATTTggAAAAACATCAgggAATTCATTTAAAgTAAATAgCTgCAAAgACCACATTggAAAgTCAATgCCAAATgTCCTAgAAgATgAAgTATATgAAACAgTTgTAgATACCTCTgAAgAAgATAgTTTTTCATTATgTTTTTCTAAATgTAgAACAAAAAATCTACAAAAAgTAAgAACTAgCAAgACTAggAAAAAAATTTTCCATgAAgCAAACgCTgATgAATgTgAAAAATCTAAAAACCAAgTgAAAgAAAAATACTCATTTgTATCTgAAgTggAACCAAATgATACTgATCCATTAgATTCAAATgTAgCACATCAgAAgCCCTTTgAgAgTggAAgTgACAAAATCTCCAAggAAgTTgTACCgTCTTTggCCTgTgAATggTCTCAACTAACCCTTTCAggTCTAAATggAgCCCAgATggAgAAAATACCCCTATTgCATATTTCTTCATgTgACCAAAATATTTCAgAAAAAgACCTATTAgACACAgAgAACAAAAgAAAgAAAgATTTTCTTACTTCAgAgAATTCTTTgCCACgTATTTCTAgCCTACCAAAATCAgAgAAgCCATTAAATgAggAAACAgTggTAAATAAgAgAgATgAAgAgCAgCATCTTgAATCTCATACAgACTgCATTCTTgCAgTAAAgCAggCAATATCTggAACTTCTCCAgTggCTTCTTCATTTCAgggTATCAAAAAgTCTATATTCAgAATAAgAgAATCACCTAAAgAgACTTTCAATgCAAgTTTTTCAggTCATATgACTgATCCAAACTTTAAAAAAgAAACTgAAgCCTCTgAAAgTggACTggAAATACATACTgTTTgCTCACAgAAggAggACTCCTTATgTCCAAATTTAATTgATAATggAAgCTggCCAgCCACCACCACACAgAATTCTgTAgCTTTgAAgAATgCAggTTTAATATCCACTTTgAAAAAgAAAACAAATAAgTTTATTTATgCTATACATgATgAAACATTTTATAAAggAAAAAAAATACCgAAAgACCAAAAATCAgAACTAATTAACTgTTCAgCCCAgTTTgAAgCAAATgCTTTTgAAgCACCACTTACATTTgCAAATgCTgATTCAggTTTATTgCATTCTTCTgTgAAAAgAAgCTgTTCACAgAATgATTCTgAAgAACCAACTTTgTCCTTAACTAgCTCTTTTgggACAATTCTgAggAAATgTTCTAgAAATgAAACATgTTCTAATAATACAgTAATCTCTCAggATCTTgATTATAAAgAAgCAAAATgTAATAAggAAAAACTACAgTTATTTATTACCCCAgAAgCTgATTCTCTgTCATgCCTgCAggAAggACAgTgTgAAAATgATCCAAAAAgCAAAAAAgTTTCAgATATAAAAgAAgAggTCTTggCTgCAgCATgTCACCCAgTACAACATTCAAAAgTggAATACAgTgATACTgACTTTCAATCCCAgAAAAgTCTTTTATATgATCATgAAAATgCCAgCACTCTTATTTTAACTCCTACTTCCAAggATgTTCTgTCAAACCTAgTCATgATTTCTAgAggCAAAgAATCATACAAAATgTCAgACAAgCTCAAAggTAACAATTATgAATCTgATgTTgAATTAACCAAAAATATTCCCATggAAAAgAATCAAgATgTATgTgCTTTAAATgAAAATTATAAAAACgTTgAgCTgTTgCCACCTgAAAAATACATgAgAgTAgCATCACCTTCAAgAAAggTACAATTCAACCAAAACACAAATCTAAgAgTAATCCAAAAAAATCAAgAAgAAACTACTTCAATTTCAAAAATAACTgTCAATCCAgACTCTgAAgAACTTTTCTCAgACAATgAgAATAATTTTgTCTTCCAAgTAgCTAATgAAAggAATAATCTTgCTTTAggAAATACTAAggAACTTCATgAAACAgACTTgACTTgTgTAAACgAACCCATTTTCAAgAACTCTACCATggTTTTATATggAgACACAggTgATAAACAAgCAACCCAAgTgTCAATTAAAAAAgATTTggTTTATgTTCTTgCAgAggAgAACAAAAATAgTgTAAAgCAgCATATAAAAATgACTCTAggTCAAgATTTAAAATCggACATCTCCTTgAATATAgATAAAATACCAgAAAAAAATAATgATTACATgAACAAATgggCAggACTCTTAggTCCAATTTCAAATCACAgTTTTggAggTAgCTTCAgAACAgCTTCAAATAAggAAATCAAgCTCTCTgAACATAACATTAAgAAgAgCAAAATgTTCTTCAAAgATATTgAAgAACAATATCCTACTAgTTTAgCTTgTgTTgAAATTgTAAATACCTTggCATTAgATAATCAAAAgAAACTgAgCAAgCCTCAgTCAATTAATACTgTATCTgCACATTTACAgAgTAgTgTAgTTgTTTCTgATTgTAAAAATAgTCATATAACCCCTCAgATgTTATTTTCCAAgCAggATTTTAATTCAAACCATAATTTAACACCTAgCCAAAAggCAgAAATTACAgAACTTTCTACTATATTAgAAgAATCAggAAgTCAgTTTgAATTTACTCAgTTTAgAAAACCAAgCTACATATTgCAgAAgAgTACATTTgAAgTgCCTgAAAACCAgATgACTATCTTAAAgACCACTTCTgAggAATgCAgAgATgCTgATCTTCATgTCATAATgAATgCCCCATCgATTggTCAggTAgACAgCAgCAAgCAATTTgAAggTACAgTTgAAATTAAACggAAgTTTgCTggCCTgTTgAAAAATgACTgTAACAAAAgTgCTTCTggTTATTTAACAgATgAAAATgAAgTggggTTTAggggCTTTTATTCTgCTCATggCACAAAACTgAATgTTTCTACTgAAgCTCTgCAAAAAgCTgTgAAACTgTTTAgTgATATTgAgAATATTAgTgAggAAACTTCTgCAgAggTACATCCAATAAgTTTATCTTCAAgTAAATgTCATgATTCTgTTgTTTCAATgTTTAAgATAgAAAATCATAATgATAAAACTgTAAgTgAAAAAAATAATAAATgCCAACTgATATTACAAAATAATATTgAAATgACTACTggCACTTTTgTTgAAgAAATTACTgAAAATTACAAgAgAAATACTgAAAATgAAgATAACAAATATACTgCTgCCAgTAgAAATTCTCATAACTTAgAATTTgATggCAgTgATTCAAgTAAAAATgATACTgTTTgTATTCATAAAgATgAAACggACTTgCTATTTACTgATCAgCACAACATATgTCTTAAATTATCTggCCAgTTTATgAAggAgggAAACACTCAgATTAAAgAAgATTTgTCAgATTTAACTTTTTTggAAgTTgCgAAAgCTCAAgAAgCATgTCATggTAATACTTCAAATAAAgAACAgTTAACTgCTACTAAAACggAgCAAAATATAAAAgATTTTgAgACTTCTgATACATTTTTTCAgACTgCAAgTgggAAAAATATTAgTgTCgCCAAAgAgTCATTTAATAAAATTgTAAATTTCTTTgATCAgAAACCAgAAgAATTgCATAACTTTTCCTTAAATTCTgAATTACATTCTgACATAAgAAAgAACAAAATggACATTCTAAgTTATgAggAAACAgACATAgTTAAACACAAAATACTgAAAgAAAgTgTCCCAgTTggTACTggAAATCAACTAgTgACCTTCCAgggACAACCCgAACgTgATgAAAAgATCAAAgAACCTACTCTgTTgggTTTTCATACAgCTAgCgggAAAAAAgTTAAAATTgCAAAggAATCTTTggACAAAgTgAAAAACCTTTTTgATgAAAAAgAgCAAggTACTAgTgAAATCACCAgTTTTAgCCATCAATgggCAAAgACCCTAAAgTACAgAgAggCCTgTAAAgACCTTgAATTAgCATgTgAgACCATTgAgATCACAgCTgCCCCAAAgTgTAAAgAAATgCAgAATTCTCTCAATAATgATAAAAACCTTgTTTCTATTgAgACTgTggTgCCACCTAAgCTCTTAAgTgATAATTTATgTAgACAAACTgAAAATCTCAAAACATCAAAAAgTATCTTTTTgAAAgTTAAAgTACATgAAAATgTAgAAAAAgAAACAgCAAAAAgTCCTgCAACTTgTTACACAAATCAgTCCCCTTATTCAgTCATTgAAAATTCAgCCTTAgCTTTTTACACAAgTTgTAgTAgAAAAACTTCTgTgAgTCAgACTTCATTACTTgAAgCAAAAAAATggCTTAgAgAAggAATATTTgATggTCAACCAgAAAgAATAAATACTgCAgATTATgTAggAAATTATTTgTATgAAAATAATTCAAACAgTACTATAgCTgAAAATgACAAAAATCATCTCTCCgAAAAACAAgATACTTATTTAAgTAACAgTAgCATgTCTAACAgCTATTCCTACCATTCTgATgAggTATATAATgATTCAggATATCTCTCAAAAAATAAACTTgATTCTggTATTgAgCCAgTATTgAAgAATgTTgAAgATCAAAAAAACACTAgTTTTTCCAAAgTAATATCCAATgTAAAAgATgCAAATgCATACCCACAAACTgTAAATgAAgATATTTgCgTTgAggAACTTgTgACTAgCTCTTCACCCTgCAAAAATAAAAATgCAgCCATTAAATTgTCCATATCTAATAgTAATAATTTTgAggTAgggCCACCTgCATTTAggATAgCCAgTggTAAAATCgTTTgTgTTTCACATgAAACAATTAAAAAAgTgAAAgACATATTTACAgACAgTTTCAgTAAAgTAATTAAggAAAACAACgAgAATAAATCAAAAATTTgCCAAACgAAAATTATggCAggTTgTTACgAggCATTggATgATTCAgAggATATTCTTCATAACTCTCTAgATAATgATgAATgTAgCACgCATTCACATAAggTTTTTgCTgACATTCAgAgTgAAgAAATTTTACAACATAACCAAAATATgTCTggATTggAgAAAgTTTCTAAAATATCACCTTgTgATgTTAgTTTggAAACTTCAgATATATgTAAATgTAgTATAgggAAgCTTCATAAgTCAgTCTCATCTgCAAATACTTgTgggATTTTTAgCACAgCAAgTggAAAATCTgTCCAggTATCAgATgCTTCATTACAAAACgCAAgACAAgTgTTTTCTgAAATAgAAgATAgTACCAAgCAAgTCTTTTCCAAAgTATTgTTTAAAAgTAACgAACATTCAgACCAgCTCACAAgAgAAgAAAATACTgCTATACgTACTCCAgAACATTTAATATCCCAAAAAggCTTTTCATATAATgTggTAAATTCATCTgCTTTCTCTggATTTAgTACAgCAAgTggAAAgCAAgTTTCCATTTTAgAAAgTTCCTTACACAAAgTTAAgggAgTgTTAgAggAATTTgATTTAATCAgAACTgAgCATAgTCTTCACTATTCACCTACgTCTAgACAAAATgTATCAAAAATACTTCCTCgTgTTgATAAgAgAAACCCAgAgCACTgTgTAAACTCAgAAATggAAAAAACCTgCAgTAAAgAATTTAAATTATCAAATAACTTAAATgTTgAAggTggTTCTTCAgAAAATAATCACTCTATTAAAgTTTCTCCATATCTCTCTCAATTTCAACAAgACAAACAACAgTTggTATTAggAACCAAAgTCTCACTTgTTgAgAACATTCATgTTTTgggAAAAgAACAggCTTCACCTAAAAACgTAAAAATggAAATTggTAAAACTgAAACTTTTTCTgATgTTCCTgTgAAAACAAATATAgAAgTTTgTTCTACTTACTCCAAAgATTCAgAAAACTACTTTgAAACAgAAgCAgTAgAAATTgCTAAAgCTTTTATggAAgATgATgAACTgACAgATTCTAAACTgCCAAgTCATgCCACACATTCTCTTTTTACATgTCCCgAAAATgAggAAATggTTTTgTCAAATTCAAgAATTggAAAAAgAAgAggAgAgCCCCTTATCTTAgTgggAgAACCCTCAATCAAAAgAAACTTATTAAATgAATTTgACAggATAATAgAAAATCAAgAAAAATCCTTAAAggCTTCAAAAAgCACTCCAgATggCACAATAAAAgATCgAAgATTgTTTATgCATCATgTTTCTTTAgAgCCgATTACCTgTgTACCCTTTCgCACAACTAAggAACgTCAAgAgATACAgAATCCAAATTTTACCgCACCTggTCAAgAATTTCTgTCTAAATCTCATTTgTATgAACATCTgACTTTggAAAAATCTTCAAgCAATTTAgCAgTTTCAggACATCCATTTTATCAAgTTTCTgCTACAAgAAATgAAAAAATgAgACACTTgATTACTACAggCAgACCAACCAAAgTCTTTgTTCCACCTTTTAAAACTAAATCACATTTTCACAgAgTTgAACAgTgTgTTAggAATATTAACTTggAggAAAACAgACAAAAgCAAAACATTgATggACATggCTCTgATgATAgTAAAAATAAgATTAATgACAATgAgATTCATCAgTTTAACAAAAACAACTCCAATCAAgCAgCAgCTgTAACTTTCACAAAgTgTgAAgAAgAACCTTTAgATTTAATTACAAgTCTTCAgAATgCCAgAgATATACAggATATgCgAATTAAgAAgAAACAAAggCAACgCgTCTTTCCACAgCCAggCAgTCTgTATCTTgCAAAAACATCCACTCTgCCTCgAATCTCTCTgAAAgCAgCAgTAggAggCCAAgTTCCCTCTgCgTgTTCTCATAAACAgCTgTATACgTATggCgTTTCTAAACATTgCATAAAAATTAACAgCAAAAATgCAgAgTCTTTTCAgTTTCACACTgAAgATTATTTTggTAAggAAAgTTTATggACTggAAAAggAATACAgTTggCTgATggTggATggCTCATACCCTCCAATgATggAAAggCTggAAAAgAAgAATTTTATAgggCTCTgTgTgACACTCCAggTgTggATCCAAAgCTTATTTCTAgAATTTgggTTTATAATCACTATAgATggATCATATggAAACTggCAgCTATggAATgTgCCTTTCCTAAggAATTTgCTAATAgATgCCTAAgCCCAgAAAgggTgCTTCTTCAACTAAAATACAgATATgATACggAAATTgATAgAAgCAgAAgATCggCTATAAAAAAgATAATggAAAgggATgACACAgCTgCAAAAACACTTgTTCTCTgTgTTTCTgACATAATTTCATTgAgCgCAAATATATCTgAAACTTCTAgCAATAAAACTAgTAgTgCAgATACCCAAAAAgTggCCATTATTgAACTTACAgATgggTggTATgCTgTTAAggCCCAgTTAgATCCTCCCCTCTTAgCTgTCTTAAAgAATggCAgACTgACAgTTggTCAgAAgATTATTCTTCATggAgCAgAACTggTgggCTCTCCTgATgCCTgTACACCTCTTgAAgCCCCAgAATCTCTTATgTTAAAgATTTCTgCTAACAgTACTCggCCTgCTCgCTggTATACCAAACTTggATTCTTTCCTgACCCTAgACCTTTTCCTCTgCCCTTATCATCgCTTTTCAgTgATggAggAAATgTTggTTgTgTTgATgTAATTATTCAAAgAgCATACCCTATACAgTggATggAgAAgACATCATCTggATTATACATATTTCgCAATgAAAgAgAgAggAAgAAAAggAAgCAgCAAAATATgTggAggCCCAACAAAAgAgACTAgAAgCCTTATTCACTAAAATTCAggAggAATTTgAAgAACATgAAgAAAACACAACAAAACCATATTTACCATCACgTgCACTAACAAgACAgCAAgTTCgTgCTTTgCAAgATggTgCAgAgCTTTATgAAgCAgTgAAgAATgCAgCAgACCCAgCTTACCTTgAgggTTATTTCAgTgAAgAgCAgTTAAgAgCCTTgAATAATCACAggCAAATgTTgAATgATAAgAAACAAgCTCAgATCCAgTTggAAATTAggAAggCCATggAATCTgCTgAACAAAAggAACAAggTTTATCAAgggATgTCACAACCgTgTggAAgTTgCgTATTgTAAgCTATTCAAAAAAAgAAAAAgATTCAgTTATACTgAgTATTTggCgTCCATCATCAgATTTATATTCTCTgTTAACAgAAggAAAgAgATACAgAATTTATCATCTTgCAACTTCAAAATCTAAAAgTAAATCTgAAAgAgCTAACATACAgTTAgCAgCgACAAAAAAAACTCAgTATCAACAACTACCggTTTCAgATgAAATTTTATTTCAgATTTACCAgCCACgggAgCCCCTTCACTTCAgCAAATTTTTAgATCCAgACTTTCAgCCATCTTgTTCTgAggTggACCTAATAggATTTgTCgTTTCTgTTgTgAAAAAAACAggACTTgCCCCTTTCgTCTATTTgTCAgACgAATgTTACAATTTACTggCAATAAAgTTTTggATAgACCTTAATgAggACATTATTAAgCCTCATATgTTAATTgCTgCAAgCAACCTCCAgTggCgACCAgAATCCAAATCAggCCTTCTTACTTTATTTgCTggAgATTTTTCTgTgTTTTCTgCTAgTCCAAAAgAgggCCACTTTCAAgAgACATTCAACAAAATgAAAAATACTgTTgAgAATATTgACATACTTTgCAATgAAgCAgAAAACAAgCTTATgCATATACTgCATgCAAATgATCCCAAgTggTCCACCCCAACTAAAgACTgTACTTCAgggCCgTACACTgCTCAAATCATTCCTggTACAggAAACAAgCTTCTgATgTCTTCTCCTAATTgTgAgATATATTATCAAAgTCCTTTATCACTTTgTATggCCAAAAggAAgTCTgTTTCCACACCTgTCTCAgCCCAgATgACTTCAAAgTCTTgTAAAggggAgAAAgAgATTgATgACCAAAAgAACTgCAAAAAgAgAAgAgCCTTggATTTCTTgAgTAgACTgCCTTTACCTCCACCTgTTAgTCCCATTTgTACATTTgTTTCTCCggCTgCACAgAAggCATTTCAgCCACCAAggAgTTgTggCACCAAATACgAAACACCCATAAAgAAAAAAgAACTgAATTCTCCTCAgATgACTCCATTTAAAAAATTCAATgAAATTTCTCTTTTggAAAgTAATTCAATAgCTgACgAAgAACTTgCATTgATAAATACCCAAgCTCTTTTgTCTggTTCAACAggAgAAAAACAATTTATATCTgTCAgTgAATCCACTAggACTgCTCCCACCAgTTCAgAAgATTATCTCAgACTgAAACgACgTTgTACTACATCTCTgATCAAAgAACAggAgAgTTCCCAggCCAgTACggAAgAATgTgAgAAAAATAAgCAggACACAATTACAACTAAAAAATATATCTAAgCATTTgCAAAggCgACAATAAATTATTgACgCTTAACCTTTCCAgTTTATAAgACTggAATATAATTTCAAACCACACATTAgTACTTATgTTgCACAATgAgAAAAgAAATTAgTTTCAAATTTACCTCAgCgTTTgTgTATCgggCAAAAATCgTTTTgCCCgATTCCgTATTggTATACTTTTgCTTCAgTTgCATATCTTAAAACTAAATgTAATTTATTAACTAATCAAgAAAAACATCTTTggCTgAgCTCggTggCTCATgCCTgTAATCCCAACACTTTgAgAAgCTgAggTgggAggAgTgCTTgAggCCAggAgTTCAAgACCAgCCTgggCAACATAgggAgACCCCCATCTTTACgAAgAAAAAAAAAAAggggAAAAgAAAATCTTTTAAATCTTTggATTTgATCACTACAAgTATTATTTTACAATCAACAAAATggTCATCCAAACTCAAACTTgAgAAAATATCTTgCTTTCAA ATTgACACATATemplate: Example 52 and 53 BCR-ABL b3a2 transcript; Full codingsequence [SEQ NO. 126]

ATggTggACCCggTgggCTTCgCggAggCgTggAAggCgCAgTTCCCggACTCAgAgCCCCCgCgCATggAgCTgCgCTCAgTgggCgACATCgAgCAggAgCTggAgCgCTgCAAggCCTCCATTCggCgCCTggAgCAggAggTgAACCAggAgCgCTTCCgCATgATCTACCTgCAgACgTTgCTggCCAAggAAAAgAAgAgCTATgACCggCAgCgATggggCTTCCggCgCgCggCgCAggCCCCCgACggCgCCTCCgAgCCCCgAgCgTCCgCgTCgCgCCCgCAgCCAgCgCCCgCCgACggAgCCgACCCgCCgCCCgCCgAggAgCCCgAggCCCggCCCgACggCgAgggTTCTCCgggTAAggCCAggCCCgggACCgCCCgCAggCCCggggCAgCCgCgTCgggggAACgggACgACCggggACCCCCCgCCAgCgTggCggCgCTCAggTCCAACTTCgAgCggATCCgCAAgggCCATggCAgCCCggggCggACgCCgAgAAgCCCTTCTACgTgAACgTCgAgTTTCACCACgAgCgCggCCTggTgAAggTCAACgACAAAgAggTgTCggACCgCATCAgCTCCCTgggCAgCCAggCCATgCAgATggAgCgCAAAAAgTCCCAgCACggCgCgggCTCgAgCgTgggggATgCATCCAggCCCCCTTACCggggACgCTCCTCggAgAgCAgCTgCggCgTCgACggCgACTACgAggACgCCgAgTTgAACCCCCgCTTCCTgAAggACAACCTgATCgACgCCAATggCggTAgCAggCCCCCTTggCCgCCCCTggAgTACCAgCCCTACCAgAgCATCTACgTCgggggCATgATggAAggggAgggCAAgggCCCgCTCCTgCgCAgCCAgAgCACCTCTgAgCAggAgAAgCgCCTTACCTggCCCCgCAggTCCTACTCCCCCCggAgTTTTgAggATTgCggAggCggCTATACCCCggACTgCAgCTCCAATgAgAACCTCACCTCCAgCgAggAggACTTCTCCTCTggCCAgTCCAgCCgCgTgTCCCCAAgCCCCACCACCTACCgCATgTTCCgggACAAAAgCCgCTCTCCCTCgCAgAACTCgCAACAgTCCTTCgACAgCAgCAgTCCCCCCACgCCgCAgTgCCATAAgCggCACCggCACTgCCCggTTgTCgTgTCCgAggCCACCATCgTgggCgTCCgCAAgACCgggCAgATCTggCCCAACgATggCgAgggCgCCTTCCATggAgACgCAgATggCTCgTTCggAACACCACCTggATACggCTgCgCTgCAgACCgggCAgAggAgCAgCgCCggCACCAAgATgggCTgCCCTACATTgATgACTCgCCCTCCTCATCgCCCCACCTCAgCAgCAAgggCAggggCAgCCgggATgCgCTggTCTCgggAgCCCTggAgTCCACTAAAgCgAgTgAgCTggACTTggAAAAgggCTTggAgATgAgAAAATgggTCCTgTCgggAATCCTggCTAgCgAggAgACTTACCTgAgCCACCTggAggCACTgCTgCTgCCCATgAAgCCTTTgAAAgCCgCTgCCACCACCTCTCAgCCggTgCTgACgAgTCAgCAgATCgAgACCATCTTCTTCAAAgTgCCTgAgCTCTACgAgATCCACAAggAgTTCTATgATgggCTCTTCCCCCgCgTgCAgCAgTggAgCCACCAgCAgCgggTgggCgACCTCTTCCAgAAgCTggCCAgCCAgCTgggTgTgTACCgggCCTTCgTggACAACTACggAgTTgCCATggAAATggCTgAgAAgTgCTgTCAggCCAATgCTCAgTTTgCAgAAATCTCCgAgAACCTgAgAgCCAgAAgCAACAAAgATgCCAAggATCCAACgACCAAgAACTCTCTggAAACTCTgCTCTACAAgCCTgTggACCgTgTgACgAggAgCACgCTggTCCTCCATgACTTgCTgAAgCACACTCCTgCCAgCCACCCTgACCACCCCTTgCTgCAggACgCCCTCCgCATCTCACAgAACTTCCTgTCCAgCATCAATgAggAgATCACACCCCgACggCAgTCCATgACggTgAAgAAgggAgAgCACCggCAgCTgCTgAAggACAgCTTCATggTggAgCTggTggAgggggCCCgCAAgCTgCgCCACgTCTTCCTgTTCACCgACCTgCTTCTCTgCACCAAgCTCAAgAAgCAgAgCggAggCAAAACgCAgCAgTATgACTgCAAATggTACATTCCgCTCACggATCTCAgCTTCCAgATggTggATgAACTggAggCAgTgCCCAACATCCCCCTggTgCCCgATgAggAgCTggACgCTTTgAAgATCAAgATCTCCCAgATCAAgAATgACATCCAgAgAgAgAAgAgggCgAACAAgggCAgCAAggCTACggAgAggCTgAAgAAgAAgCTgTCggAgCAggAgTCACTgCTgCTgCTTATgTCTCCCAgCATggCCTTCAgggTgCACAgCCgCAACggCAAgAgTTACACgTTCCTgATCTCCTCTgACTATgAgCgTgCAgAgTggAgggAgAACATCCgggAgCAgCAgAAgAAgTgTTTCAgAAgCTTCTCCCTgACATCCgTggAgCTgCAgATgCTgACCAACTCgTgTgTgAAACTCCAgACTgTCCACAgCATTCCgCTgACCATCAATAAggAAgATgATgAgTCTCCggggCTCTATgggTTTCTgAATgTCATCgTCCACTCAgCCACTggATTTAAgCAgAgTTCAAAAgCCCTTCAgCggCCAgTAgCATCTgACTTTgAgCCTCAgggTCTgAgTgAAgCCgCTCgTTggAACTCCAAggAAAACCTTCTCgCTggACCCAgTgAAAATgACCCCAACCTTTTCgTTgCACTgTATgATTTTgTggCCAgTggAgATAACACTCTAAgCATAACTAAAggTgAAAAgCTCCgggTCTTAggCTATAATCACAATggggAATggTgTgAAgCCCAAACCAAAAATggCCAAggCTgggTCCCAAgCAACTACATCACgCCAgTCAACAgTCTggAgAAACACTCCTggTACCATgggCCTgTgTCCCgCAATgCCgCTgAgTATCTgCTgAgCAgCgggATCAATggCAgCTTCTTggTgCgTgAgAgTgAgAgCAgTCCTggCCAgAggTCCATCTCgCTgAgATACgAAgggAgggTgTACCATTACAggATCAACACTgCTTCTgATggCAAgCTCTACgTCTCCTCCgAgAgCCgCTTCAACACCCTggCCgAgTTggTTCATCATCATTCAACggTggCCgACgggCTCATCACCACgCTCCATTATCCAgCCCCAAAgCgCAACAAgCCCACTgTCTATggTgTgTCCCCCAACTACgACAAgTgggAgATggAACgCACggACATCACCATgAAgCACAAgCTgggCgggggCCAgTACggggAggTgTACgAgggCgTgTggAAgAAATACAgCCTgACggTggCCgTgAAgACCTTgAAggAggACACCATggAggTggAAgAgTTCTTgAAAgAAgCTgCAgTCATgAAAgAgATCAAACACCCTAACCTggTgCAgCTCCTTggggTCTgCACCCgggAgCCCCCgTTCTATATCATCACTgAgTTCATgACCTACgggAACCTCCTggACTACCTgAgggAgTgCAACCggCAggAggTgAACgCCgTggTgCTgCTgTACATggCCACTCAgATCTCgTCAgCCATggAgTACCTggAgAAgAAAAACTTCATCCACAgAgATCTTgCTgCCCgAAACTgCCTggTAggggAgAACCACTTggTgAAggTAgCTgATTTTggCCTgAgCAggTTgATgACAggggACACCTACACAgCCCATgCTggAgCCAAgTTCCCCATCAAATggACTgCACCCgAgAgCCTggCCTACAACAAgTTCTCCATCAAgTCCgACgTCTgggCATTTggAgTATTgCTTTgggAAATTgCTACCTATggCATgTCCCCTTACCCgggAATTgACCTgTCCCAggTgTATgAgCTgCTAgAgAAggACTACCgCATggAgCgCCCAgAAggCTgCCCAgAgAAggTCTATgAACTCATgCgAgCATgTTggCAgTggAATCCCTCTgACCggCCCTCCTTTgCTgAAATCCACCAAgCCTTTgAAACAATgTTCCAggAATCCAgTATCTCAgACgAAgTggAAAAggAgCTggggAAACAAggCgTCCgTggggCTgTgAgTACCTTgCTgCAggCCCCAgAgCTgCCCACCAAgACgAggACCTCCAggAgAgCTgCAgAgCACAgAgACACCACTgACgTgCCTgAgATgCCTCACTCCAAgggCCAgggAgAgAgCgATCCTCTggACCATgAgCCTgCCgTgTCTCCATTgCTCCCTCgAAAAgAgCgAggTCCCCCggAgggCggCCTgAATgAAgATgAgCgCCTTCTCCCCAAAgACAAAAAgACCAACTTgTTCAgCgCCTTgATCAAgAAgAAgAAgAAgACAgCCCCAACCCCTCCCAAACgCAgCAgCTCCTTCCgggAgATggACggCCAgCCggAgCgCAgAggggCCggCgAggAAgAgggCCgAgACATCAgCAACggggCACTggCTTTCACCCCCTTggACACAgCTgACCCAgCCAAgTCCCCAAAgCCCAgCAATggggCTggggTCCCCAATggAgCCCTCCgggAgTCCgggggCTCAggCTTCCggTCTCCCCACCTgTggAAgAAgTCCAgCACgCTgACCAgCAgCCgCCTAgCCACCggCgAggAggAgggCggTggCAgCTCCAgCAAgCgCTTCCTgCgCTCTTgCTCCgCCTCCTgCgTTCCCCATggggCCAAggACACggAgTggAggTCAgTCACgCTgCCTCgggACTTgCAgTCCACgggAAgACAgTTTgACTCgTCCACATTTggAgggCACAAAAgTgAgAAgCCggCTCTgCCTCggAAgAgggCAggggAgAACAggTCTgACCAggTgACCCgAggCACAgTAACgCCTCCCCCCAggCTggTgAAAAAgAATgAggAAgCTgCTgATgAggTCTTCAAAgACATCATggAgTCCAgCCCgggCTCCAgCCCgCCCAACCTgACTCCAAAACCCCTCCggCggCAggTCACCgTggCCCCTgCCTCgggCCTCCCCCACAAggAAgAAgCTggAAAgggCAgTgCCTTAgggACCCCTgCTgCAgCTgAgCCAgTgACCCCCACCAgCAAAgCAggCTCAggTgCACCAgggggCACCAgCAAgggCCCCgCCgAggAgTCCAgAgTgAggAggCACAAgCACTCCTCTgAgTCgCCAgggAgggACAAggggAAATTgTCCAggCTCAAACCTgCCCCgCCgCCCCCACCAgCAgCCTCTgCAgggAAggCTggAggAAAgCCCTCgCAgAgCCCgAgCCAggAggCggCCggggAggCAgTCCTgggCgCAAAgACAAAAgCCACgAgTCTggTTgATgCTgTgAACAgTgACgCTgCCAAgCCCAgCCAgCCgggAgAgggCCTCAAAAAgCCCgTgCTCCCggCCACTCCAAAgCCACAgTCCgCCAAgCCgTCggggACCCCCATCAgCCCAgCCCCCgTTCCCTCCACgTTgCCATCAgCATCCTCggCCCTggCAggggACCAgCCgTCTTCCACCgCCTTCATCCCTCTCATATCAACCCgAgTgTCTCTTCggAAAACCCgCCAgCCTCCAgAgCggATCgCCAgCggCgCCATCACCAAgggCgTggTCCTggACAgCACCgAggCgCTgTgCCTCgCCATCTCTAggAACTCCgAgCAgATggCCAgCCACAgCgCAgTgCTggAggCCggCAAAAACCTCTACACgTTCTgCgTgAgCTATgTggATTCCATCCAgCAAATgAggAACAAgTTTgCCTTCCgAgAggCCATCAACAAACTggAgAATAATCTCCgggAgCTTCAgATCTgCCCggCgACAgCAggCAgTggTCCggCggCCACTCAggACTTCAgCAAgCTCCTCAgTTCggTgAAggAAATCAgTgACATAgTgCAgAggTAg

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1. A method of amplifying nucleic acid on a solid support, comprising:(a) providing i) a population of beads, each bead comprising one or moreamplification primers, ii) a solution of amplification reagentscomprising a thermostable polymerase, and iii) a population of nucleicacid template molecules, (b) mixing said beads and said templatemolecules in a first aliquot of said solution of amplification reagentsso as to create a mixture; (c) treating the mixture under conditionssuch that at least a portion of said template molecules non-covalentlybind to at least a portion of said beads to create bound template, andat least a portion of said primers on at least a portion of said beadsare extended by said polymerase, so as to create treated beads; (d)manipulating said treated beads so as to remove at least a portion ofsaid bound template so as to create manipulated beads; and (e)contacting said manipulated beads with a second aliquot of said solutionof amplification reagents under conditions such that at least a portionof said extended primers is amplified to create loaded beads comprisingimmobilized amplified nucleic acid and unloaded beads lacking amplifiednucleic acid.
 2. The method of claim 1, wherein said manipulating ofstep d) comprises washing said treated beads with a denaturing solution.3. The method of claim 2, wherein said denaturing solution comprisesNaOH.
 4. The method of claim 3, wherein prior to step d) between 1 and10 primers per bead are extended.
 5. The method of claim 3, whereinprior to step d) some beads comprise no extended primers.
 6. The methodof claim 1, wherein at step a) a known concentration of beads isprovided.
 7. The method of claim 6, wherein at step a) a knownconcentration of nucleic acid template molecules is provided.
 8. Themethod of claim 7, wherein at step b) the number of template moleculesto beads is less than one.
 9. The method of claim 8, wherein at step c)fewer than 50% of the beads comprise non-covalently bound template. 10.The method of claim 1, wherein the amplification primers comprise asequence which provides a code.
 11. The method of claim 10, wherein saidcode identifies the origin of the nucleic acid templates.
 12. The methodof claim 11, wherein the origin of the nucleic acid template is apatient and the code identifies the patient.
 13. The method of claim 10,wherein said code identifies the bead.
 14. The method of claim 1,wherein each bead of step (a) comprises a forward and a reverse PCRprimer.
 15. A method of amplifying nucleic acid on a solid support,comprising: a) providing i) a population of beads, each bead comprisingforward and reverse PCR primers primers, ii) a solution of amplificationreagents comprising a thermostable polymerase, and iii) a population ofnucleic acid template molecules, b) mixing said beads and said templatemolecules in a first aliquot of said solution of amplification reagentsso as to create a mixture; c) treating the mixture under conditions suchthat at least a portion of said template molecules non-covalently bindto at least a portion of said beads to create bound template, and atleast a portion of said primers on at least a portion of said beads areextended by said polymerase, so as to create treated beads; d) washingsaid treated beads with a denaturing solution so as to createmanipulated beads; and e) contacting said manipulated beads with asecond aliquot of said solution of amplification reagents underconditions such that at least a portion of said extended primers isamplified to create loaded beads comprising immobilized amplifiednucleic acid and unloaded beads lacking amplified nucleic acid.
 16. Themethod of claim 15, further comprising: (f) treating said immobilizedamplified nucleic acid so as to release at least a portion from saidloaded beads so as to create free amplified nucleic acid.
 17. The methodof claim 15, further comprising: (f) transferring at least a portion ofsaid immobilized amplified nucleic acid to a non-bead solid support. 18.The method of claim 15, wherein prior to step (a) said forward andreverse PCR primers comprised 5′ amine modifications and were attachedto agarose beads comprising a plurality of primary amine reactivefunctional groups.
 19. The method of claim 15, wherein said forward andreverse PCR primers have a region that is completely complementary to aportion of the APC gene segment
 3. 20. The method of claim 15, whereinsaid forward primer comprises a portion encoding an N-terminal epitopetag and said reverse primer comprises a portion encoding a C-terminalepitope tag.
 21. The method of claim 15, wherein said mixture is createdunder the conditions such that the ratio of the number of nucleic acidtemplate molecules to the number of beads is between 0.1:1 and 2:1. 22.The method of claim 15, wherein said mixture is created under theconditions such that the ratio of the number of nucleic acid templatemolecules to the number of beads is between 2:1 and 500, 000:1.
 23. Themethod of claim 22, wherein the ratio of the number of nucleic acidtemplate molecules to the number of beads is between 1000:1 and 100,000:1.
 24. The method of claim 22, wherein the ratio of the number ofnucleic acid template molecules to the number of beads is between10,000:1 and 100, 000:1
 25. The method of claim 22, wherein ratio of thenumber of nucleic acid template molecules to the number of beads isbetween 1000:1 and 10,000:1.
 26. The method of claim 15, wherein thepercentage of unloaded beads is between approximately 50% and 95%, asmeasured by fluorescence.
 27. The method of claim 15, wherein thepercentage of loaded beads is between approximately 1% and 5%, asmeasured by fluorescence.
 28. The method of claim 15, wherein thepercentage of loaded beads is between approximately 5% and 50%, asmeasured by fluorescence.
 29. A method of amplifying nucleic acid on asolid support, comprising: a) providing i) a population of a knownconcentration of beads, each bead comprising one or more amplificationprimers, ii) a solution of amplification reagents comprising athermostable polymerase, and iii) a population of a known concentrationof nucleic acid template molecules; b) mixing said beads and saidtemplate molecules in a first aliquot of said solution of amplificationreagents so as to create a mixture under the conditions such that theratio of the number of nucleic acid template molecules to the number ofbeads is between 1:1 and 10,000:1; c) treating the mixture underconditions such that at least a portion of said template moleculesnon-covalently bind to at least a portion of said beads to create boundtemplate, and at least a portion of said primers on at least a portionof said beads are extended by said polymerase, so as to create treatedbeads; d) manipulating said treated beads so as to remove at least aportion of said bound template so as to create manipulated beads; and e)contacting said manipulated beads with a second aliquot of said solutionof amplification reagents under conditions such that at least a portionof said extended primers is amplified to create loaded beads comprisingimmobilized amplified nucleic acid and unloaded beads lacking amplifiednucleic acid.
 30. The method of claim 29, wherein each bead of step (a)comprises a forward and a reverse PCR primer.
 31. The method of claim29, wherein said manipulating comprises washing said treated beads witha denaturing solution
 32. The method of claim 31, wherein saiddenaturing solution comprises NaOH.
 33. The method of claim 29, whereinsaid washing removes the majority of said non-covalently bound template.34. The method of claim 29, further comprising: (f) treating saidimmobilized amplified nucleic acid so as to release at least a portionfrom said loaded beads so as to create free amplified nucleic acid. 35.The method of claim 29, further comprising: (f) transferring at least aportion of said immobilized amplified nucleic acid to a non-bead solidsupport.
 36. The method of claim 30, wherein prior to step (a) saidforward and reverse PCR primers comprised 5′ amine modifications andwere attached to agarose beads comprising a plurality of primary aminereactive functional groups.
 37. The method of claim 30, wherein saidforward and reverse PCR primers have a region that is completelycomplementary to a portion of the APC gene segment
 3. 38. The method ofclaim 30, wherein said forward primer comprises a portion encoding anN-terminal epitope tag and said reverse primer comprises a portionencoding a C-terminal epitope tag.
 39. The method of claim 29, whereinthe ratio of the number of nucleic acid template molecules to the numberof beads is between 1:1 and 10:1.
 40. The method of claim 29, whereinthe ratio of the number of nucleic acid template molecules to the numberof beads is between 10:1 and 100:1
 41. The method of claim 29, whereinratio of the number of nucleic acid template molecules to the number ofbeads is between 100:1 and 1,000:1.
 42. The method of claim 29 whereinthe percentage of unloaded beads is between approximately 50% and 95%,as measured by fluorescence.
 43. The method of claim 29 wherein thepercentage of loaded beads is between approximately 1% and 5%, asmeasured by fluorescence.
 44. The method of claim 29 wherein thepercentage of loaded beads is between approximately 5% and 50%, asmeasured by fluorescence.
 45. A method of amplifying nucleic acid on asolid support, comprising: a) providing i) a population of a knownconcentration of beads, each bead comprising one or more amplificationprimers, ii) a solution of amplification reagents comprising athermostable polymerase, and iii) a population of a known concentrationof nucleic acid template molecules, b) mixing said beads and saidtemplate molecules in a first aliquot of said solution of amplificationreagents so as to create a mixture under the conditions such that theratio of the number of nucleic acid template molecules to the number ofbeads is between 0.1:1 and 2:1; c) treating the mixture under conditionssuch that at least a portion of said template molecules non-covalentlybind to at least a portion of said beads to create bound template, andat least a portion of said primers on at least a portion of said beadsare extended by said polymerase, so as to create treated beads; d)exposing said treated beads to a denaturing solution so as to createmanipulated beads; and e) contacting said manipulated beads with asecond aliquot of said solution of amplification reagents underconditions such that at least a portion of said extended primers isamplified to create loaded beads comprising immobilized amplifiednucleic acid and unloaded beads lacking amplified nucleic acid.
 46. Themethod of claim 45, wherein each bead of step (a) comprises a forwardand a reverse PCR primer.
 47. The method of claim 45, wherein saiddenaturing solution comprises NaOH and said exposing comprises at leasttwo washings of the beads.
 48. The method of claim 47, wherein saidwashings remove at least a portion of said non-covalently boundtemplate.
 49. The method of claim 47, wherein said washings removes themajority of said non-covalently bound template.
 50. The method of claim45, further comprising: (f) treating said immobilized amplified nucleicacid so as to release at least a portion from said loaded beads so as tocreate free amplified nucleic acid.
 51. The method of claim 45, furthercomprising: (f) transferring at least a portion of said immobilizedamplified nucleic acid to a non-bead solid support.
 52. The method ofclaim 45 wherein the percentage of unloaded beads is betweenapproximately 50% and 99%, as measured by fluorescence.
 53. The methodof claim 45 wherein the percentage of loaded beads is betweenapproximately 0.1% and 2%, as measured by fluorescence.